Anthony O Fedele

Valérie Carraro

Jianling Xie

Julien Averous

建凌 谢 Xie

Roger J Colbran

Christopher G Proud
email: christopher.proud@sahmri.com.

Cyclosporin A but not FK506 activates the integrated stress response in human cells

scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Cyclosporin A (CsA) is a well-established antibiotic and immunosuppressant, used in a wide range of clinical settings, including treatment of autoimmune diseases [START_REF] Mieli-Vergani | Autoimmune hepatitis[END_REF], often to prevent graft rejection in organ transplant recipients [START_REF] Van Den Bosch | Targeting the monocyte-macrophage lineage in solid[END_REF], and as therapy for lysosomal storage diseases [START_REF] Dickson | Immune tolerance improves the efficacy of enzyme replacement therapy in canine mucopolysaccharidosis I[END_REF][START_REF] Ponder | Immune response hinders therapy for lysosomal storage diseases[END_REF]. The primary mechanism by which CsA functions is by forming a complex with cyclophilin D, which in turn binds to and inhibits the activity of calcineurin, a Ca 21 /calmodulin-dependent phosphatase. CsA acts as an immunosuppressant by inhibiting the activity of calcineurin toward nuclear factor of activated T-cells (NF-AT) in Tlymphocytes. This impairs the translocation of NF-AT to the nucleus and consequently the transcription of genes for immunomodulatory factors, in particular interleukin 2 (IL-2) which is required for full T-cell activation [START_REF] Bendickova | Calcineurin-NFAT signalling in myeloid leucocytes: New prospects and pitfalls in immunosuppressive therapy[END_REF]. FK506 (also termed tacrolimus) is another structurally unrelated calcineurin inhibitor and immunosuppressant.

FK506 binds to a different immunophilin, FKBP12 [START_REF] Abraham | Immunopharmacology of rapamycin[END_REF], and may also exert additional effects independently of calcineurin [START_REF] Mori | IL-2-induced IL-5 synthesis, but not proliferation, of human CD41 T cells is suppressed by FK506[END_REF]. For instance, with respect to liver transplantation, FK506 is superior to CsA in improving survival (of patient and graft) and preventing acute rejection after liver transplantation but is also reported to increase the risk of posttransplant diabetes mellitus [START_REF] Haddad | Cyclosporin versus tacrolimus for liver transplanted patients[END_REF]. Although the complications arising from immunosuppression are an anticipated outcome of their use, other potential side effects of calcineurin inhibitors must be considered in clinical practice, particularly with respect to their nephrotoxicity [START_REF] Naesens | Calcineurin inhibitor nephrotoxicity[END_REF][START_REF] Wu | Metabolic pathway of cyclosporine A and its correlation with nephrotoxicity[END_REF].

In yeast, the phosphatase calcineurin also dephosphorylates and thereby inhibits the protein kinase Gcn2 (general control nondepressible 2), orthologous to mammalian GCN2 [START_REF] Vlahakis | A role for TOR complex 2 signaling in promoting autophagy[END_REF], which phosphorylates eukaryotic initiation factor 2 (eIF2), a key component of the protein synthesis machinery. Indeed, another study [START_REF] Rodriguez-Hernandez | The immunosuppressant FK506 uncovers a positive regulatory cross-talk between the Hog1p and Gcn2p pathways[END_REF] showed that, in yeast, FK506 activates Gcn2, further supporting the notion that inhibition of calcineurin promotes activation of Gcn2 [START_REF] Vlahakis | A role for TOR complex 2 signaling in promoting autophagy[END_REF]. Physiologically, GCN2 is activated upon amino acid starvation, which is signaled through the accumulation of uncharged tRNAs that in turn bind to and activate GCN2 [START_REF] Dong | Uncharged tRNA activates GCN2 by displacing the protein kinase moiety from a bipartite tRNA-binding domain[END_REF][START_REF] Zhang | The GCN2 eIF2a kinase is required for adaptation to amino acid deprivation in mice[END_REF][START_REF] Castilho | Keeping the eIF2 a kinase Gcn2 in check[END_REF]. GCN2 phosphorylates the a-subunit of eIF2 at serine 51 (Ser-51) [START_REF] Colthurst | Structure and regulation of eukaryotic initiation factor eIF-2. Sequence of the site in the a subunit phosphorylated by the haem-controlled repressor and by the double-stranded RNA-activated inhibitor[END_REF], thus inhibiting the activity of eIF2's guanine nucleotide-exchange factor, eIF2B, and the translation of almost all mRNAs [START_REF] Pavitt | Regulation of translation initiation factor eIF2B at the hub of the integrated stress response[END_REF]. However, translation of a subset of mRNAs, such as that for activating transcription factor (ATF) 4, is increased as an indirect consequence of eIF2a phosphorylation. ATF4 is pivotal to the integrated stress response (ISR), a process that is evoked by multiple stress stimuli, as ATF4 directly or indirectly activates genes required for cellular adaptation to stress, such as those involved in autophagy and resulting cell recovery. However, if the stress is prolonged, the ISR can promote apoptosis [START_REF] Pakos-Zebrucka | The integrated stress response[END_REF].

Gcn2 is the only eIF2a kinase in yeast, whereas in mammals there are three other eIF2 kinases [START_REF] Wek | Role of eIF2a kinases in translational control and adaptation to cellular stress[END_REF]. Expression of two of these enzymes is restricted to certain cells or conditions: protein kinase R, which is induced by type I interferons and activated by dsRNA during viral infection, and the heme-regulated eIF2a kinase (HRI), which signals iron deficiency in erythroid cells and is activated under conditions of lack of heme [START_REF] Pakos-Zebrucka | The integrated stress response[END_REF]. The third, rather widely expressed, kinase is protein kinase R-like endoplasmic reticulum kinase (PERK), which is activated as a consequence of stress in the endoplasmic reticulum (ER), such as that elicited by the accumulation of unfolded or misfolded proteins or by perturbations in the levels of certain ions in the ER lumen [START_REF] Pakos-Zebrucka | The integrated stress response[END_REF]. This contrast between yeast and mammalian cells is of interest with respect to the use of calcineurin inhibitors for immunosuppression, as several research groups have observed an association between CsA administration and increases in PERK levels but not GCN2. An increase in total PERK was observed in RPTEC/TERT1 human renal epithelial cells upon CsA treatment [START_REF] Wilmes | Application of integrated transcriptomic, proteomic and metabolomic profiling for the delineation of mechanisms of drug induced cell stress[END_REF]. Furthermore, in cervical cancerous SiHa cells, CsA induced an increase in total PERK, with a modest change in its electrophoretic migration suggesting its increased phosphorylation [START_REF] Ram | Endoplasmic reticulum vacuolation and unfolded protein response leading to paraptosis like cell death in cyclosporine A treated cancer cervix cells is mediated by cyclophilin B inhibition[END_REF]. In glioma cells, an increase in phospho-PERK (indicating its activation) was observed upon CsA treatment [START_REF] Ciechomska | Endoplasmic reticulum stress triggers autophagy in malignant glioma cells undergoing cyclosporine A-induced cell death[END_REF]. It must be noted, however, that GCN2 was not investigated in those studies. Nonetheless, these data collectively suggest a possible connection between CsA and induction of the ISR in mammalian cells, in addition to the link to GCN2 reported in yeast.

In this study, we examined whether calcineurin inhibitors promote the ISR in human and murine cells and identify the eIF2a kinases involved. We show that, surprisingly, GCN2 can also be activated by CsA and contributes to CsA's induction of the ISR. Furthermore, in stark contrast, FK506 does not elicit these effects on eIF2a phosphorylation or the ISR. Our data thus provide further insights into the distinct effects of these agents and a better understanding about the likely basis of differences in clinical toxicity.

Results

CsA induces the ISR

To assess whether, as occurs in yeast, inhibition of calcineurin could elicit activation of eIF2 kinases, we first examined the effect of the calcineurin inhibitors CsA and FK506 on eIF2a phosphorylation compared with brefeldin A (BFA), a known inducer of the ISR and ER stress [START_REF] Chardin | Brefeldin A: The advantage of being uncompetitive[END_REF][START_REF] Sano | ER stress-induced cell death mechanisms[END_REF]. To that end, the human cervical cancer HeLa and human lung carcinoma A549 cells were treated with DMSO, BFA, or either of the calcineurin inhibitors CsA or FK506 for 6 h prior to analysis of lysate samples by immunoblotting. In a similar fashion to BFA, CsA induced the phosphorylation of eIF2a at serine 51 (eIF2a P-Ser-51) (Fig. 1A) but FK506 did not. When phosphorylated on its a-subunit, eIF2 inhibits the activity of its own guanine nucleotide exchange factor, eIF2B [START_REF] Pavitt | Regulation of translation initiation factor eIF2B at the hub of the integrated stress response[END_REF]. Although this event inhibits general protein synthesis, it actually leads to enhanced translation of the mRNA encoding the transcription factor ATF4 [START_REF] Pavitt | Regulation of translation initiation factor eIF2B at the hub of the integrated stress response[END_REF][START_REF] Pakos-Zebrucka | The integrated stress response[END_REF]. Consistent with the increased phosphorylation of eIF2a P-Ser-51, CsA also increased the levels of the ATF4 protein, albeit not to the same extent as BFA (Fig. 1A). In contrast, FK506 had no effect on ATF4 protein levels, in line with its lack of effect on eIF2a phosphorylation.

To examine the effect of CsA and permit comparison with an alternative inducer of the ISR (thapsigargin, TPG), HeLa or A549 cells were exposed to BFA, CsA, or TPG for 6 h (Fig. 1B). In both lines, CsA induced increased eIF2a phosphorylation as did BFA and TPG. In summary, whereas CsA (similar with BFA or TPG) evoked the phosphorylation of eIF2a and the ISR in both cell lines tested, FK506 did not.

The compound ISRIB counters the inhibition of eIF2B by phosphorylated eIF2a, and thus its downstream effects [START_REF] Sidrauski | The small molecule ISRIB reverses the effects of eIF2a phosphorylation on translation and stress granule assembly[END_REF]. HeLa and A549 cells were treated with BFA or CsA, with or without ISRIB, prior to analysis of separate sets of lysates by im-munoblotting or by qPCR. ISRIB attenuated the induction of ATF4 by BFA and CsA in both cell lines (Fig. 2A). Additionally, ISRIB increased the phosphorylation of eIF2a, likely because GADD34, a targeting subunit for the phosphatase that acts on phosphorylated eIF2a, is normally up-regulated by increased ATF4 levels [START_REF] Harding | An integrated stress response regulates amino acid metabolism and resistance to oxidative stress[END_REF] and is thus itself inhibited by ISRIB. Finally, ATF4 drives transcription of multiple genes, including those encoding CCAAT-enhancer-binding protein homologous protein (CHOP, also a transcription factor) [START_REF] Ohoka | TRB3, a novel ER stress-inducible gene, is induced via ATF4-CHOP pathway and is involved in cell death[END_REF] and the CHOP/ ATF4 target Tribbles homologue 3 (TRB3) [START_REF] Ohoka | TRB3, a novel ER stress-inducible gene, is induced via ATF4-CHOP pathway and is involved in cell death[END_REF], among others [START_REF] Han | ER-stress-induced transcriptional regulation increases protein synthesis leading to cell death[END_REF]. Both mRNAs were markedly induced by BFA or CsA, and, as expected, these effects were reduced by ISRIB, and significantly with respect to TRB3 in HeLa cells and CHOP in A549 cells (Fig. 2B).

We also employed mouse embryonic fibroblasts (MEFs) in which the sole phosphorylation site in eIF2a (Ser-51) [START_REF] Colthurst | Structure and regulation of eukaryotic initiation factor eIF-2. Sequence of the site in the a subunit phosphorylated by the haem-controlled repressor and by the double-stranded RNA-activated inhibitor[END_REF] had been changed to alanine to preclude phosphorylation and its downstream consequences (eIF2a S51A/S51A ) [START_REF] Scheuner | Translational control is required for the unfolded protein response and in vivo glucose homeostasis[END_REF]. These cells (in addition to control MEFs) were treated with CsA for 2, 6, or 16 h. At each time point, CsA caused the up-regulation of ATF4 in WT MEFs (Fig. S1), whereas no increase in ATF4 was seen in the eIF2a S51A/S51A cells. Therefore, as expected, the phosphorylation of eIF2a is required for CsA's ability to promote the ISR, as assessed by induction of ATF4, excluding the possibility that CsA acts via other mechanisms to bring about the ISR.

In summary, consistent with BFA and TPG, CsA induced a classic stress response involving phosphorylation of eIF2a, upregulation of the expression of ATF4, and induction of ATF4target genes.

Both GCN2 and PERK contribute to induction of the ISR by CsA

As mentioned in the Introduction, several studies have indicated that CsA activates PERK [START_REF] Wilmes | Application of integrated transcriptomic, proteomic and metabolomic profiling for the delineation of mechanisms of drug induced cell stress[END_REF][START_REF] Ram | Endoplasmic reticulum vacuolation and unfolded protein response leading to paraptosis like cell death in cyclosporine A treated cancer cervix cells is mediated by cyclophilin B inhibition[END_REF][START_REF] Ciechomska | Endoplasmic reticulum stress triggers autophagy in malignant glioma cells undergoing cyclosporine A-induced cell death[END_REF], whereas in yeast, it has been demonstrated that calcineurin inhibition leads to activation of GCN2 [START_REF] Vlahakis | A role for TOR complex 2 signaling in promoting autophagy[END_REF]. On the other hand, our data (Fig. 1) demonstrate that an alternative inhibitor of calcineurin (FK506) does not activate the ISR. To assess the involvement of these two kinases in the induction of the ISR upon CsA treatment, WT, GCN2 2/2 (30), or PERK 2/2 (31) MEFs were treated with DMSO, BFA, CsA, or histidinol for 6 h (Fig. 3A). (Histidinol is an amino alcohol that inhibits the activity of the histidyl-tRNA synthetase, leading to the accumulation of uncharged tRNA His and consequent activation of GCN2.) As expected, upon BFA treatment of either WT or GCN2 2/2 MEFs, we observed a shift in the electrophoretic migration of PERK, indicative of its phosphorylation and thus its activation. In both cell lines, the activation of PERK was associated with a similar increase in ATF4 protein levels. As expected, in PERK 2/2 MEFs no significant increase in ATF4 was observed after BFA treatment (Fig. 3A). Reciprocally, histidinol treatment induced the phosphorylation of GCN2 and increased ATF4 expression only in WT and PERK 2/2 MEFs. As judged by the mobility shift, CsA evoked activation of PERK in WT and GCN2 2/2 MEFs (Fig. 3A), in line with earlier indications from glioma cells [START_REF] Ciechomska | Endoplasmic reticulum stress triggers autophagy in malignant glioma cells undergoing cyclosporine A-induced cell death[END_REF]. However, the shift appeared less pronounced with CsA treatment than with BFA.

The key observation here is that in PERK 2/2 (or GCN2 2/2 ) MEFs, as in WT cells, CsA increases ATF4 protein levels (Fig. 3A). This indicates that PERK is not the only kinase that contributes to the increase in the level of ATF4 protein caused by CsA, although it is noticeable that the increase in ATF4 upon CsA treatment was lower in GCN2 2/2 MEFs than in the two other genotypes. Although we failed to observe a significant increase in the phosphorylation of GCN2 in response to CsA, this observation does suggest a significant role of GCN2 in the induction of ATF4 protein by CsA. Intriguingly, in PERK 2/2 cells, we observed a very marked increase in the levels of phosphorylated GCN2, even under basal conditions (Fig. 3A). Moreover, it points to a potential compensatory mechanism between [START_REF] Van Den Bosch | Targeting the monocyte-macrophage lineage in solid[END_REF][START_REF] Rodriguez-Hernandez | The immunosuppressant FK506 uncovers a positive regulatory cross-talk between the Hog1p and Gcn2p pathways[END_REF], interaction P value = 0.0082; row factor F = 288.8 [START_REF] Van Den Bosch | Targeting the monocyte-macrophage lineage in solid[END_REF][START_REF] Rodriguez-Hernandez | The immunosuppressant FK506 uncovers a positive regulatory cross-talk between the Hog1p and Gcn2p pathways[END_REF], row factor P value , 0.0001; column factor F = 30.32 [START_REF] Mieli-Vergani | Autoimmune hepatitis[END_REF][START_REF] Rodriguez-Hernandez | The immunosuppressant FK506 uncovers a positive regulatory cross-talk between the Hog1p and Gcn2p pathways[END_REF], column factor P value = 0.0001. ANOVA summary for TRB3 amplification in A549 cells: Interaction F = 2.271 [START_REF] Van Den Bosch | Targeting the monocyte-macrophage lineage in solid[END_REF][START_REF] Rodriguez-Hernandez | The immunosuppressant FK506 uncovers a positive regulatory cross-talk between the Hog1p and Gcn2p pathways[END_REF], interaction P value = 0.1458; row factor F = 41.77 [START_REF] Van Den Bosch | Targeting the monocyte-macrophage lineage in solid[END_REF][START_REF] Rodriguez-Hernandez | The immunosuppressant FK506 uncovers a positive regulatory cross-talk between the Hog1p and Gcn2p pathways[END_REF], row factor P value , 0.0001; column factor F = 12.63 [START_REF] Mieli-Vergani | Autoimmune hepatitis[END_REF][START_REF] Rodriguez-Hernandez | The immunosuppressant FK506 uncovers a positive regulatory cross-talk between the Hog1p and Gcn2p pathways[END_REF], column factor P value = 0.004. For clarity, not all the significant differences are indicated.

the two kinases whereby the absence of one influences the other, a phenomenon that has previously been observed in GCN2-KO mice [START_REF] Lehman | Signaling through alternative integrated stress response pathways compensates for GCN2 loss in a mouse model of soft tissue sarcoma[END_REF]. In support of this, we also observed a large increase in total PERK protein levels in eIF2a S51A/S51A cells (Fig. S1). Further work focusing on the effects of CsA is required to unravel the mechanisms involved.

The above data clearly show that CsA induces the phosphorylation of eIF2a, an effect which can inhibit general protein synthesis. To assess the effect of CsA on protein synthesis we utilized the nonradioactive SUnSET method [START_REF] Schmidt | SUnSET, a nonradioactive method to monitor protein synthesis[END_REF], in which incorporation of puromycin into newly made proteins is assessed in a quantitative way by analyzing cell lysates by immunoblot. As shown in Fig. S2, as expected, BFA and histidinol each inhibited puromycin incorporation over a 4-h treatment period. The effect of BFA was attenuated in eIF2a S51A/S51A cells (Fig. S2), consistent with it being largely because of phosphorylation of eIF2.

The effect of histidinol was still evident in eIF2a S51A/S51A cells, likely because this agent actually causes depletion of the level of histidinyl-tRNAs, which are essential for protein synthesis, so that it still inhibits this process in the knock-in cells. The weaker effect of CsA was also retained in eIF2a S51A/S51A MEFs. Taken together, these data indicate that CsA does inhibit protein synthesis and that, although this effect is weaker than that of, e.g. BFA, it still suffices to induce ATF4. Importantly, the data also imply that CsA affects protein synthesis through additional mechanisms, not only via eIF2 phosphorylation; further work is needed to study the mechanisms involved in this.

An inhibitor of GCN2 blunts the effects of CsA

To validate a potential role for GCN2 in the effects of CsA, we made use of A92, a recently developed inhibitor of this Figure 3. CsA mediated up-regulation of ATF4 requires the eIF2a kinases GCN2 or PERK. A, protein extracts from WT, GCN2 2/2 , and PERK 2/2 MEFs that had been treated for 6 h with 1:1000 DMSO, 5 mM BFA, 10 mM CsA, or 2 mM histidinol were analyzed by immunoblotting with the indicated antibodies. B, protein extracts from WT MEFs treated for 6 h with 1:1000 DMSO, 5 mM BFA, 10 mM CsA, or 2 mM histidinol, with or without additional treatment with 5 mM A92, were immunoblotted with the indicated antibodies. C, protein extracts from PERK 2/2 MEFs treated for 6 h with 1:1000 DMSO, 5 mM BFA, 10 mM CsA, or 2 mM histidinol, with or without additional treatment with 5 mM A92 were immunoblotted with the indicated antibodies. D, total RNA was extracted from WT, GCN2 2/2 , and PERK 2/2 MEFs that had been treated for 6 h with 1:1000 DMSO or 10 mM CsA with or without additional treatment with 5 mM A92 and then evaluated via qPCR, employing primers designed to amplify fragments of the coding sequences of TRB3 and the internal control (b-actin). Error bars represent 6 S.D. Significance was evaluated using two-way ANOVA. ANOVA summary: Interaction F = 28.24 [START_REF] Dickson | Immune tolerance improves the efficacy of enzyme replacement therapy in canine mucopolysaccharidosis I[END_REF][START_REF] Colthurst | Structure and regulation of eukaryotic initiation factor eIF-2. Sequence of the site in the a subunit phosphorylated by the haem-controlled repressor and by the double-stranded RNA-activated inhibitor[END_REF], interaction P value , 0.0001; row factor F = 71.61 [START_REF] Mieli-Vergani | Autoimmune hepatitis[END_REF][START_REF] Colthurst | Structure and regulation of eukaryotic initiation factor eIF-2. Sequence of the site in the a subunit phosphorylated by the haem-controlled repressor and by the double-stranded RNA-activated inhibitor[END_REF], row factor P value , 0.0001; column factor F = 170.6 [START_REF] Dickson | Immune tolerance improves the efficacy of enzyme replacement therapy in canine mucopolysaccharidosis I[END_REF][START_REF] Colthurst | Structure and regulation of eukaryotic initiation factor eIF-2. Sequence of the site in the a subunit phosphorylated by the haem-controlled repressor and by the double-stranded RNA-activated inhibitor[END_REF], column factor P value , 0.0001. For clarity, not all the significant differences are indicated. E, protein extracts from A549 and HeLa cells treated for 6 h with 1:1000 DMSO, 5 mM BFA, 10 mM CsA, or 2 mM histidinol, with or without additional treatment with 5 mM A92, were immunoblotted with the indicated antibodies. All results are shown as the mean of the three biological replicates.

kinase [START_REF] Brazeau | Triazolo[4,5-d]pyrimidine derivatives as inhibitors of GCN2[END_REF]. As shown in Fig. 3B, A92 efficiently inhibited the phosphorylation of GCN2 and the induction of the expression of ATF4 following histidinol treatment in WT MEFs, confirming its efficacy. Remarkably, A92 also abolished the induction of ATF4 expression caused by CsA. One unexpected observation was that A92 decreased ATF4 levels in BFA-treated cells, which might, in principle, be explained by a nonspecific effect of A92 on PERK activity. Nevertheless, no effect of A92 was observed on the modest mobility shift of PERK under the different treatment conditions (Fig. 3B), suggesting it does not affect PERK. To exclude a possible effect of A92 on PERK confounding our interpretation of the CsA data, we repeated the experiment in PERK 2/2 MEFs. In these cells, we also observed that A92 abolished the increase in ATF4 expression caused by CsA (Fig. 3C), confirming that the inhibitory effect of A92 on the regulation of ATF4 by CsA is not because of interference with PERK. Finally, we also tested the impact of A92 on the level of expression of the ATF4 target gene TRB3. Following treatment with CsA for 6 h, we observed a significant increase in TRB3 mRNA, which was significantly diminished by A92 treatment in accordance with the lower levels of ATF4 shown in Fig. 3B. The results of a similar experiment with PERK 2/2 MEFs were comparable to those observed in WT MEFs, in that CsA induced an increase in TRB3 mRNA, and A92 significantly reduced this increase (Fig. 3D). Overall, these data demonstrate that, in addition to activating PERK, CsA treatment can stimulate GCN2 and thus the expression of ATF4 (as well as the induction of at least one of its target genes, TRB3). Furthermore, in both human cancer cells lines tested (HeLa and A549), A92 again strongly inhibited the induction of ATF4 by CsA (Fig. 3E). These data further show that GCN2 contributes to the effects of CsA on ATF4, i.e. the induction of the ISR.

CsA does not induce the ISR by inhibiting calcineurin

Previous data [START_REF] Rodriguez-Hernandez | The immunosuppressant FK506 uncovers a positive regulatory cross-talk between the Hog1p and Gcn2p pathways[END_REF] have shown that FK506 can induce the activation of Gcn2 in yeast [START_REF] Vlahakis | TOR complex 2-Ypk1 signaling is an essential positive regulator of the general amino acid control response and autophagy[END_REF]. However, our data from A549 cells (Fig. 1A) show that FK506, unlike CsA, did not induce phosphorylation of eIF2a or up-regulation of the ATF4 protein.

In additional experiments, FK506 did not elicit the ISR in HeLa cells (Fig. 4A), in contrast to CsA, as demonstrated by its failure to increase ATF4 protein (Fig. 4A) or CHOP mRNA (Fig. 4B) levels in both lines. Importantly, these data imply that inhibition of calcineurin is not the mechanism by which CsA induces the ISR.

In view of the lack of effect of FK506 on eIF2a phosphorylation and related responses, it was relevant to verify its efficacy confirming its ability to compete with rapamycin for binding to their common partner, the immunophilin FKBP12, because the rapamycin-FKBP12 complex inhibits the function of the mTOR complex 1 (mTORC1). Although the FK506-FKBP12 complex does not, FK506 can alleviate such inhibition and restore mTORC1 signaling [START_REF] Livi | Halcyon days of TOR: Reflections on the multiple independent discovery of the yeast and mammalian TOR proteins[END_REF]. HeLa cells were therefore exposed to a range of rapamycin concentrations (100 to 1 nM), with or without co-treatment with FK506. mTORC1 signaling was then evaluated by immunoblotting for phosphorylated ribosomal protein (rp) S6; this is an indirect target of mTORC1, because rpS6 is phosphorylated at serines 240 and 244 (P-Ser-240/244) by p70S6K, which is itself activated by phosphorylation at threonine 389 by mTORC1 [START_REF] Burnett | RAFT1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1[END_REF][START_REF] Dennis | The principal rapamycin sensitive p70(s6k) phosphorylation sites, T-229 and T-389 are differentially regulated by rapamycin insensitive kinase kinases[END_REF]. Rapamycin eliminated phosphorylation of rpS6 P-Ser-240/244, even at 1 nM, and this inhibition of mTORC1 signaling was, as expected, alleviated by FK506 (Fig. 4C), thus demonstrating its efficacy. The calcineurin inhibitor FK506 does not increase ATF4 protein or activity, whereas CsA induces markers of ER stress. A, protein extracts from HeLa and A549 cells that had been treated with 1:500 DMSO, 5 mM BFA, 10 mM CsA, or 2 mM FK506 for 24 h were immunoblotted with antibodies to ATF4 and b-actin. B, total RNA was extracted from HeLa and A549 cells that had been treated with 1:500 DMSO, 10 mM CsA, or 2 mM FK506 for 24 h for evaluation via qPCR, employing primers designed to amplify fragments of the coding sequences of CHOP and the internal control (b-actin). Error bars represent 6 S.D. Significance was evaluated using one-way ANOVA. ANOVA summary for HeLa cells: F = 53.01, P value = 0.0002. ANOVA summary for A549 cells: F = 129, P value , 0.0001. C, protein extracts from HeLa cells treated for 6 h with 1:500 DMSO or 2 mM FK506 with or without additional treatments with the indicated concentrations of rapamycin (RAPA) were immunoblotted with antibodies for total and phospho (P)-rpS6 (P-Ser-240/244) and b-actin. Results are representative of two independent experiments. D, protein extracts from HeLa and A549 cells treated with 1:1000 DMSO, 5 mM BFA, 10 mM CsA, or 2.5 mM monomethyl fumarate (MMF) for 24 h were immunoblotted with antibodies to ATF4 and b-actin. Results are representative of two independent experiments. E, protein extracts from A549 cells that had been treated with 1:1000 DMSO, 5 mM BFA, 10 mM CsA, or 2 mM FK506 for 6 h were immunoblotted with the indicated antibodies. Results are representative of two independent experiments. F, total RNA was extracted from HeLa and A549 cells treated with 1:1000 DMSO, 5 mM BFA, or 10 mM CsA for 6 h. This was used as a template for the preparation of cDNA, which was analyzed by qualitative PCR, employing primers (42) designed to amplify fulllength (442 bp) and IRE1-processed (416 bp) fragments of the coding sequences of XBP1. All results are represented as the mean of the three biological replicates, unless otherwise stated.

CsA does not appear to induce the ISR through mitochondrial stress

Given that our data suggest that CsA may exert its effects independently of inhibiting calcineurin (otherwise FK506 would elicit similar reactions) our attention was drawn to another binding partner for CsA, cyclophilin D. Cyclophilin D forms part of the mitochondrial permeability transition pore whose opening enhances the permeability of the mitochondrial membrane and is associated with induction of mitochondrial stress [START_REF] Briston | Mitochondrial permeability transition: A molecular lesion with multiple drug targets[END_REF]. We speculated that CsA may exert its effects on the ISR by binding to and ablating cyclophilin D functions. To mimic this effect of CsA without using that compound, we performed siRNA-mediated knockdown of CYPD. However, this caused no reproducible change in the modulation of ATF4 protein (Fig. S3), indicating that impairment of cyclophilin D function does not induce the ISR.

The ISR has recently been suggested to be a consequence of the mitochondrial stress response in mammals. The enzyme implicated in this effect ( 40) is mitochondrial fumarate hydratase (encoded by the FH gene in humans), which catalyzes the conversion of fumarate to L-malate. It was shown that either knockdown of this mRNA or treatment of cells with monomethyl fumarate (to simulate the increase in fumarate resulting from reduced FH expression or function) increased the expression of ATF4 and CHOP [START_REF] Quirós | Multi-omics analysis identifies ATF4 as a key regulator of the mitochondrial stress response in mammals[END_REF]. To assess whether CsA could be inducing the ISR by causing mitochondrial stress and, in turn, interfering with fumarate hydratase, we repeated the latter experiment. Interestingly, however, in our hands supplementing the cells' growth medium with monomethyl fumarate did not itself elicit the expected increase of ATF4 protein in either HeLa or A549 cells, unlike BFA or CsA (Fig. 4D). Finally, to assess further the possibility that CsA-induced mitochondrial stress accounts the activation of the ISR, ATP levels were measured using the CellTiter-Glo ® Luminescent Cell Viability Assay. If CsA induced mitochondrial stress in our experiments, reduced ATP levels would be anticipated. Data were normalized to the number of viable cells as determined by calcein AM staining subsequent to treatment with vehicle, CsA, or FK506. However, according to this assay, they were not significantly altered by either CsA or FK506 (Fig. S4). Thus, collectively, our data do not indicate that CsA induces the ISR through mitochondrial stress.

CsA also activates the IRE1 arm of the unfolded protein response

Immunoblotting of A549 and HeLa cells treated with either BFA, CsA, or FK506 to detect BiP/grp78 demonstrated that CsA (like BFA), but not FK506, increased the expression of BiP, a chaperone protein which functions in the ER, indicating that it elicits an ER stress response in these cell lines (Fig. 4E). Indeed, in addition to activating PERK, ER stress also affects the splicing of the mRNA encoding XBP1, a transcription factor that plays an important role in the unfolded protein response (which in turn is triggered by ER stress) [START_REF] Vlahakis | TOR complex 2-Ypk1 signaling is an essential positive regulator of the general amino acid control response and autophagy[END_REF] and whose activation involves splicing of the coding sequence of its mRNA by IRE1 [START_REF] Huang | Emerging roles for the ER stress sensor IRE1a in metabolic regulation and disease[END_REF]. To study this, total RNA was extracted from HeLa and A549 cells that had been treated with DMSO, BFA, or CsA for 6 h and then analyzed by qualitative RT-PCR employing primers designed to amplify full-length (442 bp) and IRE1-processed (416 bp) fragments of the coding sequence of XBP1 [START_REF] Yoshida | XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor[END_REF]. In both lines, both BFA and CsA treatment increased splicing of the XBP1 RNA, again indicating activation of the unfolded protein response (Fig. 4F).

Discussion

Our data indicate that the calcineurin inhibitor CsA activates both GCN2 and PERK in mammals (Fig. 5). However, strikingly, FK506 did not elicit the same effects, which strongly implies that inhibition of calcineurin itself is not responsible for the effects of CsA on these kinases.

As mentioned previously, associations between calcineurin activity and ISR inhibition have been suggested by studies in both yeast and vertebrates. With respect to the former, calcineurin's phosphatase activity has been implicated in the inhibition of Gcn2 [START_REF] Vlahakis | A role for TOR complex 2 signaling in promoting autophagy[END_REF]. Additionally, several research groups have previously observed increases in total and/or phospho-PERK levels (and, by implication, ER stress) following administration of the calcineurin inhibitor CsA [START_REF] Wilmes | Application of integrated transcriptomic, proteomic and metabolomic profiling for the delineation of mechanisms of drug induced cell stress[END_REF][START_REF] Ram | Endoplasmic reticulum vacuolation and unfolded protein response leading to paraptosis like cell death in cyclosporine A treated cancer cervix cells is mediated by cyclophilin B inhibition[END_REF][START_REF] Ciechomska | Endoplasmic reticulum stress triggers autophagy in malignant glioma cells undergoing cyclosporine A-induced cell death[END_REF], although no mechanistic information was provided and other eIF2 kinases were not examined.

Physiologically, GCN2 is activated consequent to reduced intracellular amino acid levels via binding of uncharged tRNA [START_REF] Castilho | Keeping the eIF2 a kinase Gcn2 in check[END_REF]. It is possible that CsA interacts directly with GCN2 in a similar way to that of uncharged tRNA. Recently, an inhibitor of GCN2 (termed A92) has been developed [START_REF] Brazeau | Triazolo[4,5-d]pyrimidine derivatives as inhibitors of GCN2[END_REF], and we have demonstrated that this inhibited the effect of CsA on the ISR in a range of cell lines (Fig. 3, B-E)

On the other hand, the ability of CsA to activate PERK seems unlikely to be because of a specific direct effect on PERK itself, as CsA also induced another arm of the unfolded protein response, i.e. the altered splicing of XBP1 (Fig. 4, E andF). Indeed, the up-regulation of the ER-localized chaperone BiP/ grp78 (an activator of both PERK and XBP1 splicing, which is up-regulated upon ER stress) potentially implies an ability for CsA to interfere with BiP/grp78. This suggests that CsA promotes ER stress or a related process. Indeed, some previous reports have hinted at effects of CsA (but not FK506) on ER stress, as indicated by increased expression of the ER proteins BiP/GRP78 and IRE1a, alongside cellular vacuolation in cervical cancer SiHa cells [START_REF] Ram | Endoplasmic reticulum vacuolation and unfolded protein response leading to paraptosis like cell death in cyclosporine A treated cancer cervix cells is mediated by cyclophilin B inhibition[END_REF]. The authors concluded that the differing effects were not because of inhibition of calcineurin, which is consistent with our observations that they are not evoked by FK506. Furthermore, CsA, which has also been implicated in posttransplantation diabetes mellitus [START_REF] Haddad | Cyclosporin versus tacrolimus for liver transplanted patients[END_REF], has been shown to promote XBP1 splicing in pancreatic b-cells, although interestingly neither ATF4 nor BiP/grp78 was affected [START_REF] Kim | Rosiglitazone protects the pancreatic b-cell death induced by cyclosporine A[END_REF]. Finally, certain ER proteins are also reported to be elevated in children who had received CsA and developed nephrotic syndrome [START_REF] Hama | Endoplasmic reticulum stress with low-dose cyclosporine in frequently relapsing nephrotic syndrome[END_REF], supporting potential clinical relevance of our findings. Our data imply that proteins induced by ER stress may be valuable as biomarkers of CsA toxicity. Our data also imply that CsA inhibits protein synthesis by additional mechanisms, not only by inducing the phosphorylation of eIF2a.

An unexpected outcome of this work was the observations made in derivatives of MEFs where either PERK or GCN2 had been removed, or where the regulatory phosphorylation site in eIF2a (Ser-51) has been mutated. In PERK 2/2 cells, we observed a large increase in the levels of phosphorylated GCN2 (Fig. 3A), whereas in eIF2a S51A/S51A cells there was a dramatic increase in total PERK protein levels, even under basal conditions (Fig. S1). This indicates novel, uncharacterized links between these components of the ISR, whereby the absence of one of them may cause increases in another, perhaps as a feedback mechanism to compensate for the deficiency. Not only does this indicate bidirectional crosstalk through the ISR pathway, but also suggests that even in normal conditions, there is a basal level of ISR activity which, no matter how low, is still crucial to cellular function. This should form the basis of future studies.

In summary, the major findings of our study include the first evidence that CsA can evoke increased phosphorylation of eIF2a and downstream ISR through either GCN2 or PERK. Furthermore, although both act as immunosuppressants by inhibiting calcineurin activity, our data demonstrate that CsA elicits eIF2-mediated stress responses in mammalian cells but FK506 does not. This strongly suggests that CsA's mode of action in these phenomena is not by inhibiting calcineurin. Importantly, as FK506 does not activate these stress responses, this difference likely helps to explain its lesser side effects as compared with CsA, resulting in better tolerance and outcomes in patients who have undergone transplant surgery.

Experimental procedures

Chemicals for cell treatments

All chemicals were purchased from Sigma-Aldrich. Brefeldin A (BFA), rapamycin, CsA, FK506, thapsigargin (TPG), and ISR inhibitor (ISRIB, integrated stress response inhibitor) [START_REF] Sidrauski | The small molecule ISRIB reverses the effects of eIF2a phosphorylation on translation and stress granule assembly[END_REF] were each dissolved in DMSO, while histidinol dihydrochloride, puromycin, and monomethyl fumarate were prepared in Milli-Q water.

Cell culture A549 cells derived from cancerous lung tissue, HeLa cells derived from cervical cancer cells, and all genotypes of mouse embryonic fibroblasts were maintained in Dulbecco's modified Eagle's medium with 10% (v/v) fetal bovine serum (Life Technologies) and 100 units/ml penicillin/streptomycin (Sigma-Aldrich) and incubated at 37°C and 5% (v/v) CO 2 .

RNA extraction

Cells grown in 6-well plates were washed using 2 ml PBS, followed by the addition of 500 ml TRIzol Reagent (Thermo Fisher). Total RNA was then prepared according to the manufacturer's instructions and provided a template for complementary DNA (cDNA) preparation.

Qualitative RT-PCR cDNA was first synthesized from total RNA using the Super-Script III First-Strand Synthesis System (Life Technologies). Fifty ml reactions were prepared using cDNA (prepared with 6.25 ng starting RNA), 100 nM forward and reverse primers, 1 unit HotStarTaq DNA polymerase, 13 Q-solution, 1 mM MgCl 2 , 200 mM dNTP mix, and Milli-Q water. Reactions were performed at 95°C for 15 min; 35 times (94°C for 1 min; 60°C for 1 min; 72°C for 1 min); 72°C for 10 min. Previously published oligonucleotide primers [START_REF] Yoshida | XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor[END_REF] were employed to amplify a portion of the coding sequence of XBP1 and flanking the 26 nucleotides removed by IRE1.

Quantitative real time PCR (qPCR)

cDNA was first prepared from total RNA using the Super-Script III First-Strand Synthesis System (Life Technologies). Oligonucleotide primers designed to amplify fragments of the coding sequences of the genes of interest (Table 1) and the internal control, b-actin. To each well were added 0.843 SYBR Green Fast Mix (Applied Biosystems), 5-10 ml cDNA (6.25-12.5 ng starting RNA), 200 nM forward and reverse primers and Milli-Q water to 20 ml. qPCR reactions proceeded as follows on an ABI Step One Plus qPCR instrument (Applied Biosystems): 95°C for 20 s; 40 times (95°C for 3 s; 60°C for 30 s). The comparative threshold cycle protocol was employed to determine amounts of the target mRNA.

Immunoblotting

Cells grown in 6-well plates were washed using 2 ml ice-cold PBS, then lysed with 100-300 ml RIPA buffer supplemented with 2.5 mM Na 2 H 2 P 2 O 7 (Sigma-Aldrich), 1 mM b-glycerophosphate (Sigma-Aldrich), 1 mM Na 3 VO 4 (Sigma-Aldrich), and 13 Protease Inhibitor mixture (Roche). Cells were then rocked for 30 min at 4°C, scraped, and transferred to a microcentrifuge tube. Cell suspensions were centrifuged at 15,871 3 g for 30 min at 4°C, then the supernatant was maintained and transferred to new microfuge tubes. Protein concentration was quantified for sample normalization using the Lowry procedure [START_REF] Lowry | Protein measurement with the Folin phenol reagent[END_REF].

Equal amounts (by protein) of samples were subjected to SDS-PAGE, then transferred to a nitrocellulose membrane. The membrane was blocked for ;90 min in PBS containing 0.1% Tween 20 and 2% (w/v) BSA at 4°C. Membranes were then rolled overnight at 4°C in the same solution supplemented with one of the following antibodies: 1/1000 rabbit polyclonal anti-ATF4 (Cell Signaling Technology); 1/1000 mouse monoclonal anti-eIF2a (Cell Signaling Technology); 1/1000 rabbit polyclonal anti-eIF2a P-S51 (Cell Signaling Technology); 1/ 1000 rabbit monoclonal anti-BiP (Cell Signaling Technology); rabbit monoclonal anti-GCN2 P-T899; 1/1000 (Abcam); 1/ 1000 rabbit monoclonal anti-GCN2 (Cell Signaling Technology); 1/5000 mouse monoclonal anti-puromycin clone 12D10 (Millipore), or 1/10000 mouse monoclonal anti-b-actin (Sigma-Aldrich). The following day, the membrane was washed three times for 5 to 10 min at room temperature in PBS containing 0.1% Tween 20, then incubated at room temperature for 1 h in PBS containing 0.1% Tween 20 and 2% BSA plus 1/3000 horseradish peroxidase-linked sheep anti-mouse (GE Healthcare), goat anti-rabbit (Millipore), or 1/5000 mouse antihuman b-actin (Sigma-Aldrich) IgG. The membrane was then washed three times for 5 to 10 min at room temperature in PBS with 0.1% Tween 20, drained, treated with the SuperSignal West Pico Plus Chemiluminescent Substrate (Thermo Fisher), developed on a LAS-4000 apparatus (Fujifilm), and analyzed using Multigauge 1D software (Fujifilm). In some cases, further densitometric quantification was implemented using ImageJ (National Institutes of Health).

CellTiter-Glo assay ATP levels of cells grown in 96-well plates were determined via the application of a CellTiter-Glo ® Luminescent Cell Viability Assay and normalized to viable cell number as determined via calcein AM, according to the manufacturer's instructions (Promega).

Measure of protein synthesis by SUnSET

SUnSET experiments were performed as described previously [START_REF] Schmidt | SUnSET, a nonradioactive method to monitor protein synthesis[END_REF], briefly cells were treated with 5 mg/ml of puromycin during the last 30 min of the experiment. Then, the cells were harvested, and immunoblotting was performed using TGX FastCast Stain-Free Acrylamide 10% gel (Bio-Rad). Stain-free imaging allows for the normalization of the puromycin signal to the total protein on a blot.

Statistics

Figure 1 .

 1 Figure 1. CsA increases levels of ATF4 protein and activity. A, protein extracts from HeLa and A549 cells treated with 1:500 DMSO, 5 mM BFA, 10 mM CsA, or 2 mM FK506 for 6 h were immunoblotted with the indicated antibodies. B, protein extracts from HeLa and A549 cells treated with 1:1000 DMSO, 5 mM BFA, 10 mM CsA, or 1 mM TPG for 6 h were immunoblotted with the indicated antibodies. All results are representative of three independent experiments.

Figure 2 .

 2 Figure 2. ISRIB inhibits the up-regulation ATF4 by CsA. A, protein extracts from HeLa and A549 cells treated with 1:1000 DMSO, 5 mM BFA or 10 mM CsA for 6 h, with or without 2 mM ISRIB, were immunoblotted using the indicated antibodies. Results are representative of three independent experiments. B, total RNA was extracted from similarly treated HeLa and A549 cells for evaluation via qPCR, employing primers designed to amplify fragments of the coding sequences of CHOP and TRB3 and the internal control (b-actin). The data are the mean of the three biological replicates. Error bars represent 6 S.D. Significance was evaluated using two-way ANOVA. ANOVA summary for CHOP amplification in HeLa cells: Interaction F = 18.79 (2, 12), interaction P value = 0.0002; row factor F = 158.2 (2, 12), row factor P value , 0.0001; column factor F = 35.72 (1, 12), column factor P value , 0.0001. ANOVA summary for TRB3 amplification in HeLa cells: Interaction F = 51.84 (2, 12), interaction P value , 0.0001; row factor F = 133.2 (2, 12), row factor P value , 0.0001; column factor F = 175.1 (1, 12), column factor P value , 0.0001. ANOVA summary for CHOP amplification in A549 cells: Interaction F = 7.354[START_REF] Van Den Bosch | Targeting the monocyte-macrophage lineage in solid[END_REF][START_REF] Rodriguez-Hernandez | The immunosuppressant FK506 uncovers a positive regulatory cross-talk between the Hog1p and Gcn2p pathways[END_REF], interaction P value = 0.0082; row factor F = 288.8[START_REF] Van Den Bosch | Targeting the monocyte-macrophage lineage in solid[END_REF][START_REF] Rodriguez-Hernandez | The immunosuppressant FK506 uncovers a positive regulatory cross-talk between the Hog1p and Gcn2p pathways[END_REF], row factor P value , 0.0001; column factor F = 30.32[START_REF] Mieli-Vergani | Autoimmune hepatitis[END_REF][START_REF] Rodriguez-Hernandez | The immunosuppressant FK506 uncovers a positive regulatory cross-talk between the Hog1p and Gcn2p pathways[END_REF], column factor P value = 0.0001. ANOVA summary for TRB3 amplification in A549 cells: Interaction F = 2.271[START_REF] Van Den Bosch | Targeting the monocyte-macrophage lineage in solid[END_REF][START_REF] Rodriguez-Hernandez | The immunosuppressant FK506 uncovers a positive regulatory cross-talk between the Hog1p and Gcn2p pathways[END_REF], interaction P value = 0.1458; row factor F = 41.77[START_REF] Van Den Bosch | Targeting the monocyte-macrophage lineage in solid[END_REF][START_REF] Rodriguez-Hernandez | The immunosuppressant FK506 uncovers a positive regulatory cross-talk between the Hog1p and Gcn2p pathways[END_REF], row factor P value , 0.0001; column factor F = 12.63[START_REF] Mieli-Vergani | Autoimmune hepatitis[END_REF][START_REF] Rodriguez-Hernandez | The immunosuppressant FK506 uncovers a positive regulatory cross-talk between the Hog1p and Gcn2p pathways[END_REF], column factor P value = 0.004. For clarity, not all the significant differences are indicated.

Figure 4 .

 4 Figure 4. The calcineurin inhibitor FK506 does not increase ATF4 protein or activity, whereas CsA induces markers of ER stress. A, protein extracts from HeLa and A549 cells that had been treated with 1:500 DMSO, 5 mM BFA, 10 mM CsA, or 2 mM FK506 for 24 h were immunoblotted with antibodies to ATF4 and b-actin. B, total RNA was extracted from HeLa and A549 cells that had been treated with 1:500 DMSO, 10 mM CsA, or 2 mM FK506 for 24 h for evaluation via qPCR, employing primers designed to amplify fragments of the coding sequences of CHOP and the internal control (b-actin). Error bars represent 6 S.D. Significance was evaluated using one-way ANOVA. ANOVA summary for HeLa cells: F = 53.01, P value = 0.0002. ANOVA summary for A549 cells: F = 129, P value , 0.0001. C, protein extracts from HeLa cells treated for 6 h with 1:500 DMSO or 2 mM FK506 with or without additional treatments with the indicated concentrations of rapamycin (RAPA) were immunoblotted with antibodies for total and phospho (P)-rpS6 (P-Ser-240/244) and b-actin. Results are representative of two independent experiments. D, protein extracts from HeLa and A549 cells treated with 1:1000 DMSO, 5 mM BFA, 10 mM CsA, or 2.5 mM monomethyl fumarate (MMF) for 24 h were immunoblotted with antibodies to ATF4 and b-actin. Results are representative of two independent experiments. E, protein extracts from A549 cells that had been treated with 1:1000 DMSO, 5 mM BFA, 10 mM CsA, or 2 mM FK506 for 6 h were immunoblotted with the indicated antibodies. Results are representative of two independent experiments. F, total RNA was extracted from HeLa and A549 cells treated with 1:1000 DMSO, 5 mM BFA, or 10 mM CsA for 6 h. This was used as a template for the preparation of cDNA, which was analyzed by qualitative PCR, employing primers (42) designed to amplify fulllength (442 bp) and IRE1-processed (416 bp) fragments of the coding sequences of XBP1. All results are represented as the mean of the three biological replicates, unless otherwise stated.

Figure 5 .

 5 Figure 5. CsA activates both GCN2 and PERK in mammalian cells.

  Abbreviations-The abbreviations used are: CsA, cyclosporin A; FK506, tacrolimus; NF-AT, nuclear factor of activated T-cells; ISR, integrated stress response; eIF, eukaryotic initiation factor; ATF,

Table 1

 1 Oligonucleotides employed in this study for PCR ER, endoplasmic reticulum; PERK, protein kinase R-like ER kinase; TPG, thapsigargin; MEF, mouse embryonic fibroblast; rp, ribosomal protein; BFA, brefeldin A; cDNA, complementary DNA; ANOVA, analysis of variance.

	Gene	Accession	Human/Mouse	Forward (59!39)	Reverse (59!39)
	CHOP	NM_004083.5	H	CAGCTGAGTCATTGCCTTTC	TTGATTCTTCCTCTTCATTTCC
	TRB3	NM_021158.4	H	GTATACCTGCAAGGTGTACC	AAAAAGGCGTAGAGGAGCTG
	XBP1 (42)	NM_005080	H	CCTTGTAGTTGAGAACCAGG	GGGGCTTGGTATATATGTGG
	TRB3	NM_175093.2	M	CCAGAGATACTCAGCTCCCG	GAGGAGACAGCGGATCAGAC
	b-actin			CTGGCACCACACCTTCTAC	GGGCACAGTGTGGGTGAC

J. Biol. Chem. (2020) 295(44) 15134-15143 15135

J. Biol. Chem. (2020) 295(44) 15134-15143 15137

J. Biol. Chem. (2020) 295(44) 15134-15143 15139

J. Biol. Chem. (2020) 295(44) 15134-15143 15141

J. Biol. Chem. (2020) 295(44) 15134-15143 15143

Acknowledgments-We are grateful to Dr. Xuemin Wang, Stuart De Poi, Roman Lenchine, and Lauren Sandeman (Lifelong Health Theme, SAHMRI) for advice and technical assistance. We thank Professor Terence Herbert (Lincoln, UK), Dr. Anne-Catherine Maurin (Clermont-Ferrand, France), Dr. Randal Kaufman (La Jolla, CA, USA), and Professor David Ron (Cambridge, UK) for providing MEFs from genetically modified mice.

Funding and additional information-This work was supported by

Data availability

All data of this publication have been contained within this manuscript or are available from the corresponding author (Chris Proud; SAHMRI, Adelaide, Australia; E-mail: Christopher. Proud@sahmri.com) upon reasonable request.


domain: info spi

text:
Samuel Marie-Rose

Giovanny Arbelaez
email: arbelaez5@univ-lorraine.fr

Mauricio Camargo

A referential framework for the choice of prototypes in the design phase of a mobile application

Keywords: Referential framework, Prototyping, Design phase, Mobile application I

This paper aims at providing designers a referential framework for the choice of the most appropriate prototype to use to test a specific aspect of a mobile application during the design phase. In order to reach this objective, a literature review was carried out for characterizing and identifying the different types of prototypes. Four main features were distinguished for characterizing prototypes: level of fidelity, sustainability, medium and interactivity. After this review, a focus was done on the design phase of a mobile application and the referential framework was proposed by defining eight aspects commonly tested while designing. Then, a case study helped to test the framework in order to validate its usefulness and consistency.

INTRODUCTION

Prototyping has received much attention for many years due to the major role it plays in the design phase of a product [START_REF] Camburn | A Systematic Method for Design Prototyping[END_REF]. A prototype is an artifact that imitates one or more features of a product, service or system [START_REF] Wood | Product design : techniques in reverse engineering and new product development[END_REF]. It is really useful in the design process because it allows having a concrete idea of the final product. However, due to the various types of prototypes that exist, it could be difficult to choose the most appropriate one, to test a specific aspect of a product during its design phase. One way to help designers to select the most appropriate prototype to use is to define features that allow characterizing the different types of prototypes and there have been various researches regarding that. For example, Baccino [START_REF] Baccino | Prototypage[END_REF] proposed to characterize prototypes by using four features: level of fidelity, interactivity, sustainability and medium. Furthermore, more recently [START_REF] Camburn | A Systematic Method for Design Prototyping[END_REF] provided guidelines for incorporating prototyping in the design process. Nevertheless, although features were defined for prototypes, they did not use it to characterize each one. Moreover, little attention has been paid to the classification/selection of the most appropriate prototype to use for testing a specific aspect of a product during the design phase. The present paper presents a referential framework for selecting such a prototype. Based on a literature review, it describes the four features that characterize a prototype before providing a set of prototypes usually used. It then proposes a table that characterizes each type of prototype before providing support for selecting the most appropriate prototype to use for testing a specific aspect of a product in the design phase of a mobile application.

II. LITERATURE REVIEW

A prototype is defined as an artifact that imitates one or more features of a product, service or system [START_REF] Wood | Product design : techniques in reverse engineering and new product development[END_REF]. Prototyping has been key for designing products and solutions. Henry Ford used several prototypes before finalizing the model T design [START_REF] Womack | The Machine That Changed the World: The Story of Lean Production--Toyota's Secret Weapon in the Global Car Wars That Is Now Revolutionizing World Industry[END_REF]. This highlights that in addition to being a process included in the design phase of a product/service, prototyping can be used in various fields (manufacturing, marketing, automotive industry or others). Literature provides much information about when and how to integrate prototyping into the design process. Depending on the desired objective, a specific prototype will be used. During the design phase, it's needed to take numerous factors into account to produce a prototype. Prototypes are assessed on the following features: testing, timing, ideation, feedback and fidelity. Testing: Although every test includes some cost, effort and time to be produced [START_REF] Dahan | An Extreme-Value Model of Concept Testing[END_REF], a separating feature between a prototype and a design concept is the possibility to realize some tests and validate design hypothesis. In a design process, it's important to collect information to move forward in product development without spending a lot of money and time. At this point, prototyping appears to be a response to this need. Timing: It is widely recognized that the timing of prototyping is important in the design process. Research conducted on prototyping in the early stages of the design phase argued for early and frequent prototyping as a way to test ideas early [START_REF] Kelley | The art of innovation: lessons in creativity from IDEO, America's leading design firm[END_REF] and to help build confidence in product development in a team [START_REF] Gerber | Prototyping: Facing uncertainty through small wins[END_REF]. Decisions made in a good timing of a design process have great bearing on the outcome of the design in terms of design success, cost of manufacturing and time to market [START_REF] Häggman | The Influence of Timing in Exploratory Prototyping and Other Activities in Design Projects[END_REF] Ideation: When exploring concepts, prototypes are essential. They enable organic learning and discovery [START_REF] Gill | Prototypes as Inquiry, Visualization and Communication[END_REF] in addition to enabling ideas to be more functional [START_REF] Viswanathan | Build to Learn: Effective Strategies to Train Tomorrow's Designers[END_REF] Feedback: It is important to get feedback at different stages of the design process. This is why prototyping is useful to give to stakeholders, peers and others an idea of the final product and detect possible problems. Fidelity: The level of fidelity of a prototype must depend on the objective of this prototype. Higher fidelity representations lead to more accurate interpretation, comments and feedback by third parties [START_REF] Hannah | A user study of interpretability of engineering design representations[END_REF].

While the previously described characteristics are taken into account when defining a prototype, after the literature analysis, it was found that there are mainly four characteristics that are defined before deciding the type of prototype to use when designing a mobile application. Those characteristics are: sustainability, interactivity, level of fidelity and medium.

A. Sustainability

A prototype can be throwaway, evolutionary or incremental. Each one has a particular objective. A throwaway prototype aims at developing a small part of the system and using it to enable end users to try it out and evaluate it [START_REF] Baccino | Prototypage[END_REF]. The user provides feedback which can be quickly incorporated into the product development. This cheap and fast prototype is then discarded. An evolutionary prototype is a robust prototype built in a structured manner and constantly refined (c.f. Fig. 1). The artifact, when built, forms the core of the new system and the improvements and further requirements will be then implemented based on the feedback collected [START_REF] Christie | Prototyping Strategies: Literature Review and Identification of Critical Variables[END_REF]. An incremental prototype aims at building and testing progressively the designed product as separate prototypes. At the end, these prototypes are merged in an overall design.

B. Interactivity

A prototype may be horizontal or vertical. A horizontal prototype demonstrates only the outer layer of the human interface, such as windows, options and screens. This does not enable direct interaction with the user. This type of prototype is used to check that users understand the structure and functionalities of the product but also to clarify its scope and requirements. A vertical prototype also called interactive prototype, allows the user to interact with the system [START_REF] Baccino | Prototypage[END_REF]. It is an enhanced complete elaboration of a single subsystem or function. This type of prototype is useful for refining database design, collecting information on interface needs and clarifying complex requirements.

C. Level of fidelity

A prototype is also characterized by its level of fidelity (c.f. Table 1). The concept of fidelity indicates how closely the prototypes resemble the finished product in terms of interaction, visual appearance, data model and level of detail [START_REF] Walker | High-Fidelity or Low-Fidelity, Paper or Computer? Choosing Attributes when Testing Web Prototypes[END_REF]. Prototypes are usually classified as low-fidelity or high fidelity, but to better describe more recent prototyping approaches, two other classifications has been proposed: mixed-fidelity [START_REF] Mccurdy | Breaking the Fidelity Barrier: An Examination of Our Current Characterization of Prototypes and an Example of a Mixedfidelity Success[END_REF] and multi-fidelity [START_REF] Coyette | Multi-fidelity Prototyping of User Interfaces[END_REF].

1) Low-fidelity A low-fidelity prototype is sketchy and incomplete. It consists of a series of drafts representing scenarios and flows [START_REF] Rudd | Low vs. High-fidelity Prototyping Debate[END_REF]. It aims at quickly producing the model and broadly testing concepts. This cheap prototyping occurs early in the design process and allow detecting needs of the users and basic usability problems [START_REF] Nissinen | User experience prototyping : a literature review[END_REF].

2) High-fidelity High-fidelity prototypes are interactive, usually computerbased and present the complete functionality of a service or product. The similarities of this type of prototype and the final product are so high in terms of details and functionality, that users may not note the difference [START_REF] Nissinen | User experience prototyping : a literature review[END_REF]. Furthermore, it is more expensive and time-consuming than low-fidelity prototypes and is usually used at the end of the design process [START_REF] Baccino | Prototypage[END_REF]. -More expensive to develop -Time-consuming to create -Inefficient for proof-ofconcept designs -Not effective for requirements gathering.

3) Mixed-fidelity

Mixed-fidelity prototyping combines low-fidelity and highfidelity prototyping in a single prototype. It mixes static layouts and interactive features. It allows designers to focus on a specific interface issue, by exploring and making refinements at higher-fidelity and leaving other elements at lower-fidelity to keep them within the context of the overall design [START_REF] Petrie | Mixed-fidelity prototyping of user interfaces[END_REF]. Its advantages and disadvantages would depend on the chosen combination of the fidelity levels.

D. Medium

A prototype can be represented in three different mediums: paper, electronic and video.

1) Paper prototyping Paper prototyping is a technique in which paper models are used to create and test user interfaces in an early stage of a design process. It is a quick and cheap way to validate the value of ideas and concepts. Users can have a concrete idea of the main options of the product or service by using these prototypes.

2) Electronic prototyping An electronic prototype provides a screen layout that can be static or interactive. It aims at creating a user interface that looks and behaves like the final product. Electronic prototyping can be carried out once the overall flows and functionality have been agreed. This iterative design approach is the next step after paper prototyping.

3) Video prototyping Video prototypes use video to illustrate the user interactions with the system. In this case, the user does not interact directly with the system, but can have a good idea of the system use. It is usually used with a focus group or to complete a paper prototype.

Table 2 presents how appropriate it is to use one specific medium for a prototype regarding the already described characteristics.

Paper Electronic Video Throwaway R R R Iterative R R NA Incremental NA R NA Horizontal R R NA Vertical M R R Low-fidelity R M M Mixed-fidelity NA R R High-fidelity NA R M NA: Not Applicable; M: Marginal; R: Recommended

Types of prototypes

While many types of prototypes exist, we found in the literature review ten of the most commonly used: -Video prototype -Feasibility prototype developed to determine the feasibility of various solutions. It is used to resolve technical risks related to the development in terms of performance, compatibility of components for instance. -Vertical prototype -Horizontal prototype -Rapid prototyping is a technique of quickly creating a scale model of a part or finished product using a threedimensional computer-aided design -Simulation prototype is digitally creating a physical product to predict its performance in the real world -Storyboard is a series of drawings or images that describes a product, service or system in the form of a story. It aims at determining useable sequences for presenting information -Wireframes are presented in the form of illustrations that capture a specific aspect of design such as an idea, layout or sequence -Animations are drawn images put in a sequence to present a scenario -A mock-up provides an overall visual of the product Depending on what designers are working on, they will select the most appropriate prototype to use. In a design process, when prototyping, it will be useful to determine which specific aspect to test, and after, to choose the prototype that will allow collecting more feedbacks and help to improve the designed product.

The literature review allows having a clear vision of the different types of prototypes existing but also to understand the features that characterizes these prototypes (c.f. Fig. 2).

Through the review, it is interesting to note that there are links between features. For instance, most of horizontal prototypes are throwaway and are often low-fidelity. We can also remark that most of electronic prototypes are vertical (interactive). Furthermore, based on the literature review, it is interesting to highlight a gap in the characterization of each type of prototype. In fact, at our knowledge, we have no found publications characterizing each prototype and providing information about in which forms the prototype come in. That is why it appears important, in this paper, to propose a table presenting the features of each prototype (c.f. TABLE 3). To achieve this, eight of the ten prototypes presented in the literature review have been selected to be characterized. Based on this review, an analysis aiming at understanding the features of each prototype has been done. TABLE 3 presents the results of this analysis and interpretation. The table can be useful for a designer or a team when choosing a prototype to produce that must involve specific features. For instance, a team which wants to produce an artifact involving the following features: mixed-fidelity, throwaway, horizontal (static) and video, can use the table to note that the most appropriate model to produce is the video prototype. Then, this team can easily select this model to realize the desired test. Another example, by using TABLE 3, a mobile application designer who wants to produce a model involving the following features: high-fidelity, vertical (interactive) and electronic, can note that two types of prototypes seem to be appropriate. These artifacts are the feasibility prototype and simulation prototype. However, these two models are different given that they allow realizing distinct tests. Indeed, while feasibility prototype allows testing technical aspects and compatibility between components for a mobile application, simulation prototype allows testing user interactions with the application. This shows the need to introduce the type of test criteria to help designers in selecting prototype to produce depending on the test to realize. Here is the second contribution of this research aiming at facilitating the choice of the most appropriate prototype to use to test a specific aspect of a mobile application during the design phase.

The present paper focuses its research on mobile applications.

III. REFERENTIAL FRAMEWORK FOR THE CHOICE OF PROTOTYPES IN THE DESIGN PHASE OF A MOBILE APPLICATION

In order to support designers in their choice of a prototype for testing a specific aspect of a mobile application, it was critical to distinguish three main development focus of a mobile application in the design phase: the user journey, the valueadded services ideas and the technical challenges. The user journey presents the different layouts and the sequence that will face the customer while using the application. This development focus is critical for allowing customers to interact with the application and for involving them in strategy work [START_REF] Folstad | Living Labs for innovation and development of information and communication technology: a literature review[END_REF]. The value-added services ideas of the mobile application are the development focus that aims at determining which services will be developed for adding value to the standard services. When designing the product, it is important to gauge the level of interest of a service idea with the user, to evaluate if it is interesting to develop it. These services depend on the main objective of the application. Finally, the technical challenges refer to the technical obstacles necessary to master in order to implement the mobile application. With this three-development focus, eight aspects usually tested during the design phase of a mobile application have been distinguished:

-Functional aspect: that aims at imitating the functions of the final product as closely as possible no matter how different they look from the final product. It allows testing the coherence of functionalities proposed and the ease of use of the system. -Aesthetic aspect: is a factor that plays an important role in user experience. This aspect is closely linked to usability. Users perceive more aesthetic interfaces as much more intuitive than those considered to be less aesthetically pleasing. Indeed, the visual appearance of the application has a positive impact on performance leading to reduced task completion times [START_REF] Sonderegger | The influence of design aesthetics in usability testing: Effects on user performance and perceived usability[END_REF]. The level of aesthetics is mainly determined by colour combination, visual layout and text front of the interface [START_REF] Lee | The Impact of Cognitive Style on User Preference Based on Usability and Aesthetics for Computer-Based Systems[END_REF]. It is critical to test this aspect to ensure that the interface is easy and pleasant to use. -Technical feasibility: seeks to explore the technical constraints of complex design features. It allows to study what is feasible or not from a technical and technological point of view (data sharing, data storage or others) -Interaction: refers to the interaction between the user and the system. It is the ability of the system to respond to a specific command issued by the user -Interoperability: is the ability of different information technology systems and software applications to exchange data, communicate and use the information that has been exchanged. During interoperability testing, we check how the data from an application/system is transferred into another application/system and further processed to give the accepted output. -Content: refers to the information presented at each stage of the use of the application -Usage scenario: refers to the different stages faced by the user to activate and use a service when using the mobile application -User experience: refers to a person's attitudes and emotions while interacting with a particular system or service [START_REF] Hassenzahl | Hedonic, Emotional, and Experiential Perspectives on Product Quality[END_REF], [START_REF] Hassenzahl | User experience -a research agenda[END_REF].

Each aspect is important to test when designing a mobile application in order to measure and assess the level of interest of the user for the product. Moreover, by realizing these tests, designers ensure the design of a consistent and feasible application that matches the needs and expectations of customers. Table 4 is a proposal of a referential framework for the choice of prototypes in the design phase of a mobile application. To use it, the designer must refer to the development focus matter (user journey, value-added services ideas or technical challenges) and after, depending on the aspect to test, he can choose the prototype proposed by the table. In the case of various options, designers can use some factors such as costs or prototyping time to support its choice. In order to experiment and test the proposed referential framework proposed, we used it in the design phase of a multi-device mobile application realized by a French telecommunications company. This application aims at allowing users to manage their connected objects from their mobile.

The framework was applied on two development focus of the mobile application designed: user journey and value-added services ideas. Indeed, Table 4 was a good support for choosing the most appropriate prototype to use for each test to realize. Furthermore, when choosing, some factors such as costs and prototyping time was important to take into account. TABLE 5 presents the prototypes chosen for each development phase and aspect to test when designing. The following sub-sections provide details about the choices done during the design phase of the mobile application.

A. User journey prototyping

The first design stages of the application were to develop the management of connected objects inventory and the activation of the telecommunications company services (payments, communication, subscription and content).

The objective was to realize a user journey that allowed customers to add their connected objects on the application, to subscribe to an appropriate offer for each device and to manage these objects. It was also important to develop this journey for value-added services management.

In order to properly design the user journey, it was useful to imagine a concrete case. That is why we imagined the journey for the adding and management of a connected suitcase. Therefore, it was critical to propose various journeys in order to assess and design the adding phase of the suitcase on the application, the payment phase and the stage of management of the connected object. Fig. 3 shows an example of wireframes sequences developed for the user journey.

The aim of this prototype was to test five aspects of the journey: functional, aesthetics, interactivity, content and user experience. Based on the referential framework (c.f. Table 4), wireframes were used with the software Balsamiq for testing the functional, aesthetics, content aspects and the user experience. This prototype allowed visualizing and testing the consistency of the application functionalities, its architecture, the visual quality, the use sequence, the difficulties that users can face while using the application, the clarity of the content but also the customer behaviour regarding the design of the system. During this prototyping phase, several iterations were realized in order to improve and enrich the customer journey.

Here is one of the advantages of wireframes that allows realizing many iterations before designing a prototype similar to the final solution and that meets the established requirements.

The next steps of the user journey design will aim at testing the technical feasibility of the solutions previously developed.

B. Prototyping of value-added services ideas

In order to meet the needs of connected objects users and to avoid designing an application that only allows adding and managing devices, it appeared important to identify valueadded services that can allow customers to really take advantage of their connected objects inventory.

To develop and select ideas of services, Design Thinking was used. The first step aimed at identifying and understanding needs, expectations and difficulties of the users regarding the use and management of their connected objects. After, it was critical to define the profile of customers interested in the application before generating value-added services ideas. The following step aimed at prototyping these ideas by producing storyboards to test it with users. Design Thinking method was structured with 5 phases: Empathize -Define -Ideate -Prototype -Test.

The storyboards were essential to test the ideas with users. They allowed collecting feedback on the user experience, assessing the coherence of the usage scenario and testing the level of interest of the customers for the ideas. Furthermore, it is important to note that two factors played a role in the choice of this type of prototype to use. Indeed, the low cost and low prototyping time were factors that encouraged using storyboards besides video prototype and animations (c.f. Table 4).

For the prototyping phase, storyboards were produced to clearly illustrate ideas of services generated during the ideation phase. This type of prototype was appropriate to facilitate the illustration and presentation of the ideas, to assess the user experience regarding these ideas but also to illustrate the usage scenario proposed. Fig. 4 shows a storyboard produced to illustrate an idea and to be tested with users. Each storyboard is composed of three drawings and texts to describe the scenario imagined. This research aimed at proposing a referential framework for the choice of prototypes in the design phase of a mobile application. Firstly, a literature review provided us with an outlook of the work done in previous research on prototyping and their features. On this review, we distinguished four main features of prototypes: level of fidelity, sustainability, medium and interactivity. In addition to these features, ten types of prototypes commonly used have been identified. A table characterizing each one of these prototypes have been proposed. Furthermore, this allowed us to propose a referential framework for supporting designers in their choice of the most appropriate prototype to use to test a specific aspect of a mobile application during the design phase. Then, a case study was carried out to test this framework and two of three development focus were tested.

In order to go further with this research, it could be interesting to develop and explore three points:

• Although eight types of prototypes were identified for testing different aspects, some other models could be interesting and adapted for testing these aspects. In order to enrich the contribution, we could investigate other prototypes and analyse their benefits and features to determine which aspects they could allow to test

• Furthermore, for proposing the referential framework (c.f. Table 4), eight aspects to test during the design phase of a mobile application were distinguished. However, we could identify more aspect for testing. Depending on the project and the designed application, it could be interesting to test other aspects that could help designers to improve the system. Further research could aim at investigating these aspects. • Finally, in order to better support design teams, we could propose a detailed list of existing tools available for each type of prototype and related to digital products development (for example, we can use Balsamiq, Axure RP or Sketch to develop wireframes. To go deeper, we could describe each tool, present its advantages and disadvantages and then indicate when it could be interesting to use it.

TABLE 1 .

 1 ADVANTAGES AND DISADVANTAGES OF LOW-FIDELITY AND HIGH-FIDELITY PROTOTYPES BASED ON[START_REF] Rudd | Low vs. High-fidelity Prototyping Debate[END_REF]

Fig. 1 .

 1 Fig. 1. Steps of an evolutionary prototype

Fig. 2 .

 2 Fig. 2. The four main features of prototypes

  Author's accepted version. For final version see: S. Marie-Rose, G. Arbelaez and M. Camargo, "A referential framework for the choice of prototypes in the design phase of a mobile application," 2019 IEEE International Conference on Engineering, Technology and Innovation (ICE/ITMC), Valbonne Sophia-Antipolis, France, 2019, pp. 1-7, doi: 10.1109/ICE.2019.8792568.

Fig. 4 .Fig. 3 .

 43 Fig. 4. Example of storyboard describing an idea to be tested with users Fig. 3. A sequence of wireframes of the user journey

TABLE 2 .

 2 DEGREE OF APPLICABILITY OF THE MEDIUM REGARDING THE TYPE OF PROTOTYPE BASED ON[START_REF] Baccino | Prototypage[END_REF]

TABLE 3 .

 3 CHARACTERIZATION OF EACH PROTOTYPE

Features Prototype Video prot. Feasibility prototype Rapid prot. Simulation prototype Storyboard Wireframe Animations Mock-up

		Paper					x	x x
	Medium	Electronic		x x x	x
		Video	x				x	x
	Interactivity	Horizontal x Vertical	x	x	x	x x x x x
		Throwaway x		x x x x x x
	Sustainability	Evolutionary		x			x
		Incremental		x
		Low-fidelity					x	x x
	Level of	Mixed-	x		x		x
	fidelity	fidelity
		High-fidelity		x		x

TABLE 4 .

 4 REFERENTIAL FRAMEWORK FOR THE CHOICE OF PROTOTYPES IN THE DESIGN PHASE OF A MOBILE APPLICATION

		Development focus
		User	Value
	Aspect to test	journey of	added	Technical
		mobile	service	challenges
		application	ideas
	Functional	Wireframe	NA	NA
	Aesthetic	Wireframe	NA	NA
	Technical
	feasibility

TABLE 5

 5 presents the prototypes used during the design phase of the mobile application based on the referential framework.

TABLE 5 .

 5 PROTOTYPES USED DURING THE DESIGN PHASE OF THE MOBILE APPLICATION BASED ON THE REFERENTIAL FRAMEWORK PROPOSED

		Development focus
	Aspect to test	User journey	Value added service ideas
	Functional	Wireframe via Balsamiq	NA
	Aesthetic	Wireframe via Balsamiq	NA
	Technical feasibility	Not tested	Not tested
	Interaction	Simulation prototype (chatbot)	NA
	Interoperability	Not tested	NA
	Content	Wireframe via Balsamiq	NA
	Usage scenario	Wireframe via Balsamiq	Storyboard
	User experience	Wireframe via Balsamiq	Storyboard
	V. CONCLUSION AND PERSPECTIVES

ACKNOWLEDGMENT S.M.R would like to thank Orange Group and the whole team coordinated by Sébastien Bertrand (at the Direction Connected Objects & Partnership), who made possible the participation to the design of a mobile application.