Laura Blanco

Hadj Hassen

Michel Tijani

Aurélien Noiret

Martín L Blanco

A new experimental and analytical study of fully grouted rockbolts

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

INTRODUCTION

Rockbolts have been extensively used for the past 30 years as reinforcement elements. A rockbolt consists of a bar inserted in a borehole that is drilled into the surrounding soil or rock mass and anchored to it by means of a fixture. A rockbolt reinforcement system has four principal components [START_REF] Windsor | Rock reinforcement-technology, testing, design and evaluation[END_REF]: the rock or soil, the reinforcing bar, the internal fixture to the borehole wall and the external fixture to the excavation surface (a plate and a nut in most cases). Such system is very efficient if used in one or several of the following applications [START_REF] Chappell | Rockbolts and shear stiffness in jointed rock mass[END_REF][START_REF] Fine | Le soutènement des galeries minières[END_REF];

• Stabilization of blocky rock masses, provided the far end of the bolt is anchored to a stable zone; • Rock confinement, contributing to the use of the broken rock belt to confine the stable rock mass; • Improvement of the mechanical properties of the rock mass.

In addition, the easy installation and low cost of rockbolts compared to those of other reinforcement elements have contributed to their worldwide success [START_REF] Stillborg | Professional Users Handbook for Rock Bolting[END_REF].

Fully grouted rockbolts are able to support tensile, compressive, shear and bending loads. The current study focuses on their tensile behavior because it is very often encountered and furthermore it allows studying the load transfer mechanism between the rock mass and the reinforcement element. Experience throughout the world has shown that under tensile solicitations failure often takes place by debonding at either the bolt-grout interface or at the grout-rock interface, depending on which one is weaker: in fact, if a bolted rock mass tries to move, a load will be progressively transferred to the rod and a shear stress will develop consequently along the embedded length (see figure 1). As the shear strength of the interface is progressively reached, debonding will occur.

Since Freeman [START_REF] Freeman | The behaviour of fully-bonded rock bolts in the Kielder experimental tunnel[END_REF] monitored for the first time in the 1970s the loading process and the stress distribution along the embedded length of a fully grouted rockbolt, numerous studies aiming at a better understanding of the load transfer mechanism between the surrounding ground and the bar have been conducted. Such studies comprise field monitoring [START_REF] Choquet | Development and field testing of a tension measuring gauge for cable bolts used as ground support[END_REF][START_REF]Guidance on the use of cable bolts to support roadways in coal mines[END_REF], laboratory [START_REF] Benmokrane | Laboratory Evaluation of Cement-Based Grouts and Grouted Rock Anchors[END_REF][START_REF] Moosavi | Bond of cement grouted reinforcing bars under constant radial pressure[END_REF][START_REF] Hagan | Variation in load transfer of a fully encapsulated rockbolt[END_REF][START_REF] Hyett | A Constitutive Law for Bond Failure of Fully-grouted Cable Bolts Using a Modified Hoek Cell[END_REF] and in situ testing [START_REF] Hyett | The Effect of Rock Mass Confinement on the Bond Strength of Fully Grouted Cable Bolts[END_REF][START_REF]Coal mine roadway support system handbook[END_REF][START_REF] Chen | Mechanical behavior of the bamboo-steel composite rock-bolt[END_REF][START_REF] Rong | Testing study on working mechanism of fully grouted bolts of thread steel and smooth steel[END_REF], analytical approaches [START_REF] Hyett | A Constitutive Law for Bond Failure of Fully-grouted Cable Bolts Using a Modified Hoek Cell[END_REF][START_REF] Farmer | Stress Distribution along a Resin Grouted Rock Anchor[END_REF][START_REF] Kaiser | Bond Strength of Grouted Cable Bolts[END_REF][START_REF] Li | Analytical models for rock bolts[END_REF][START_REF] Ren | An analytical analysis of the full range behaviour of grouted rockbolts based on a tri-linear bond-slip model[END_REF] and numerical modelling [START_REF] Larson | Bolt action in jointed rock[END_REF][START_REF] Fuller | Bolt load simulation and its practical application[END_REF][START_REF] Ivanović | Modelling of debonding along the fixed anchor length[END_REF]. They have undoubtedly contributed to a better comprehension of the mechanical behavior of grouted rockbolts, to better design techniques and to a reduction in costs. Nevertheless, some aspects such as the analytical prediction of the axial load and the axial slip generated in an anchored rockbolt and the influence of parameters like the bolt profile or the confining pressure need to be reviewed.

The work currently undertaken by the authors tries to improve the knowledge acquired through these past works. The bond-slip model and the basis of the new analytical solution are presented first. The new experimental bench designed to test fully grouted rockbolts and cable bolts is described later, and the results obtained on steel deformed bars and Fibre Reinforced Polymer (FRP) rockbolts are discussed. So far, the influence on the anchoring capacity of the confining pressure, the embedment length, the type and profile of the bolt and the quality of the grouting material has been investigated. From these results, a constitutive law for the failure interface is being constructed. At the same time, in situ pull-out tests [START_REF] Andra | Essais de traction boulons d'ancrage[END_REF] performed in ANDRA's (

THE BOND-SLIP MODEL

The bond-slip model is the constitutive law of the interface between the rockbolt and the grouting material (or between the grouting material and the rock mass). It represents the local shear stress -shear slip relationship, τ(W), of the failure interface; thus it takes into account the debonding mechanism that occurs as the shear slip increases. A bond-slip model often consists of three parts: elasticity (i.e., the interface is totally coupled), plasticity (a decoupling front starts to propagate from the point where the load is applied to the free end of the bolt) and a residual phase, that remains due to friction.

Such a constitutive law can be derived from pull-out test results, provided the tests are conducted under accurate conditions. At laboratory scale, this means that the embedment length should be short enough to ensure a uniform distribution of the shear stress along the anchored length (i.e., the shear slip is the same at every point of the rockbolt). Since a rheological law is not dependent upon the length, it can be validated by conducting two pull-out tests under identical conditions but using two different embedment lengths. This fact has been taken into account in the experimental study presented in section 4.

The rheological characterisation of the joint interface proves to be interesting in the design of rockbolt reinforcement patterns (bolt dimensions and spacing, maximum and residual strengths and associated slips).

The stress distribution in an elementary bolt length dz is shown in figure 1. As it can be inferred from the diagram, knowledge of the interface constitutive law allows predicting the load on the rockbolt according to Eq. ( 1):

dZ W R dF ) ( 2 τ π = (1)
Regarding numerical modelling, such law permits a discretized approach, in contrast to classic homogenisation and reinforced-properties (namely, cohesion and friction angle of the rock mass) approaches.

ANALYTICAL APPROACH

A mathematical model able to predict the full-range behavior of a fully grouted rockbolt (in terms of axial force and axial displacement) subjected to a tensile load has been recently developed. It is limited to monotonic loading, which means that the axial displacement of every point of the bar must be an increasing function of time. The data needed for its application are: bolt radius R b and length L, bolt's Young modulus E b and the constitutive law of the rockbolt-grout (or rock-grout) joint interface, τ(W). The system equations and the resolution method are explained in detail in [START_REF] Blanco Martín | A New Analytical Solution to the Mechanical Behaviour of Fully Grouted Rockbolts Subjected to Pull-out Tests[END_REF], where a complete development of the solution in the case of a tri-linear bond-slip model is also included. Therefore, only the most important features are mentioned here.

Let W(Z,T) be the axial displacement of the rockbolt that is in front of point Z of the joint interface. The axial deformation of the rockbolt is

Z W W ∂ ∂ = '
. The reference axis Z is fixed on the borehole wall, so that Z=0 corresponds to the far end of such joint and Z=L corresponds to the point where the load is applied. The position Z=kT (where k=0 if the bolt length is supposed infinite and k=1 otherwise) corresponds to the free end of the bolt throughout the pull-out process. At this point, the displacement is T.

Two hypotheses have been considered in the model:

• The bolt remains in the elastic range during the whole loading process; • The relative displacement (shear slip) between the rockbolt and the grouting material equals the axial displacement of the rockbolt (i.e., the axial deformation of the bolt is negligible).

Then, the constitutive equations of the rockbolt and the interface joint are respectively:

' 2 2 W E R R F b b b b π σ π = = (2) ) (W S = τ (3)
Combining the equilibrium Eq. (1) with Eqs. ( 2) and (3) the governing equation of the problem is:

) ( 2 ' ' W S R E W b b = (4)
The problem consists therefore of finding the axial displacement W (as a function of Z) that solves Eq. ( 4). Boundary conditions are:

0 ) , ( '
) , ( = = T kT W T T kT W (5)
According to Eq. ( 2), F is calculated from W'; thus the axial response of the rockbolt during a pull-out test is completely characterised by W'(L,T) and W(L,T).

Compared to previous solutions [START_REF] Farmer | Stress Distribution along a Resin Grouted Rock Anchor[END_REF][START_REF] Li | Analytical models for rock bolts[END_REF][START_REF] Ren | An analytical analysis of the full range behaviour of grouted rockbolts based on a tri-linear bond-slip model[END_REF] the innovation of the current approach consists in using boundary conditions that only concern the free end of the bolt (in the previous cases, one boundary condition concerned the point where the load is applied). The control parameter of the new model is the displacement T of this end.

The validation of this new approach has been carried out by comparing experimental results issued from both field and laboratory pull-out tests with the forcedisplacement relationship predicted by the new model. The results are in good agreement, as shown in figures 2a and 2b. The in situ test was performed on a 15.26 mm diameter threaded steel rockbolt grouted using epoxy resin. The embedment length was 5 m. A tri-linear bondslip model was assumed for the bolt-grout interface.

Regarding the laboratory situation, a 20 mm diameter deformed steel rockbolt was anchored over 100 mm using Lokset resin grout. The confining pressure was 5 MPa. In this case, a nonlinear function τ(W) including an exponential decay proved to give better results than a classic tri-linear function. Further comparisons will be provided in the next section.

EXPERIMENTAL STUDY

In order to clarify the mechanism of load transfer between the ground and the rockbolt, pull-out tests have been carried out within the context of this research program. They have been conducted both in the laboratory and in situ, and an attempt to relate the results (in terms of peak and residual shear strengths and slips) has been made.

Laboratory investigation: equipment and procedure

A new experimental bench has been designed and recently put into operation. Previous works [START_REF] Benmokrane | Laboratory Evaluation of Cement-Based Grouts and Grouted Rock Anchors[END_REF][START_REF] Moosavi | Bond of cement grouted reinforcing bars under constant radial pressure[END_REF][START_REF] Hagan | Variation in load transfer of a fully encapsulated rockbolt[END_REF][START_REF] Hyett | A Constitutive Law for Bond Failure of Fully-grouted Cable Bolts Using a Modified Hoek Cell[END_REF][START_REF]Testing and standards for rock reinforcement consumables[END_REF] have been considered during the design stage, so that improvements could be introduced in order to overcome some of the limitations encountered in the past. The main features of this new bench are described below.

The bench allows studying not only fully grouted rockbolts but also grouted cable bolts. Different grouting materials (resin, cementitious grouts) can be studied. The influence of the following parameters may be investigated: the confining pressure, the embedment length, the quality of the grout, the nature (type, profile) of the bolt, the roughness of the borehole surface, the influence of the thickness of the grout annulus and the rock sample tested.

Figure 3 shows a cutaway section of the current bench.

The confining pressure may be maintained constant or varied throughout the pull-out test; as a matter of fact, if the volume of confining oil is kept unchanged during the axial slip of the rockbolt, the effect of radial dilation can be studied. The axial force is applied via a 950 kN capacity & 70 mm stroke hollow ram jack and the axial displacement is measured using three LVDT sensors (the mean value is withheld). The force is measured using a 350 kN customized load cell. The tests are carried out under displacement control, using a constant displacement rate of 0.02 mm/s. Data are captured using LabVIEW. The acquisition frequency is 5 Hz. In order to study the residual capacity of the system (post-peak response), the bolts are normally pulled up to 70 mm.

As it can be seen in figure 3, the bench is based upon the double embedment principle: the bar length embedded in the rock mass represents the length where a debonding process might take place in a real situation, whereas the bolt length fixed to the metallic tube accounts for the fully anchored, load-carrying part. Thus in laboratory situations, no relative slip should take place inside the metallic tube. A highly resistant resin-based grout is used to anchor the rod to this tube.

It is important to note that, due to design considerations, the reaction force is made directly on the rock mass. As a result of the stress regime in the rock sample, a crater depression is likely to appear around the point where the bolt protrudes from the borehole. A comparison between tests where the crater formation took place and tests where it was prevented is presented in the following subsection. Since the main purpose of laboratory pullout tests is to derive a constitutive law for the failure interface, such an end effect should be avoided in order to simplify the analysis and the interpretation of the results. For this reason, a metallic plate whose internal diameter matches the diameter of the tested bar has been designed. Once the constitutive law is determined, the tests where the crater depression occurred can be used to validate the new law by means of numerical modelling.

In the current research program, sandstone has been selected to be used as rock sample because of its good mechanical properties (see table 1), its homogeneity and also because it is a rock often present in coal mining environments (rockbolts and cable bolts are common support elements in gateroads for longwall mining).

One of the principal innovations of this bench consists in avoiding the well-known rotation phenomenon, especially in the case of cable bolts. Because of their low torsional rigidity due to their design (6 or more wires wound around a central kingwire), cables are likely to rotate under tensile loads. In fact, as the cable is progressively pulled out, it tends to "unscrew" from the grout imprints, which could cause the rock sample to rotate as well. To solve this matter, two actions have been undertaken: to prevent relative rotation between the rock sample and the jack, three dowel pins are inserted in three boreholes 10 mm long drilled in the rock sample, the end plate and the upper piston. At the same time, two dowels are inserted between the piston of the jack and the threaded plate, so that any possible relative displacement between the metallic tube and the hollow ram jack is blocked.

Laboratory investigation: results

So far, 29 pull-out tests have been performed. Two types of rockbolts have been studied: steel HA25 deformed bars and FRP rockbolts. The nominal diameter of both rods is d b =25 mm and their Young's moduli are 160,000 and 40,000 MPa respectively. The lateral surface of both bolts is ribbed: FRP rockbolts present a continuous standard thread (pitch length, l=10 mm; height of the asperities, h=1.5 mm), whereas HA25 bars are provided with a series of non-continuous annular ribs. The lateral surface of this bar is divided in two parts, so that one half presents parallel indentations and in the other half the ridges are laid following two different inclinations.

On average, l=16 mm and h=1.7mm. Figure 4 shows both bars. Six different confining pressures have been tested in order to take account of stress variation in field situations. Three anchoring lengths have been used: 90, 130 and 150 mm, which correspond to 3.6d b , 5.2d b and 6.0d b respectively. This choice has been recommended by other authors [START_REF] Benmokrane | Laboratory Evaluation of Cement-Based Grouts and Grouted Rock Anchors[END_REF][START_REF] Moosavi | Bond of cement grouted reinforcing bars under constant radial pressure[END_REF][START_REF] Hagan | Variation in load transfer of a fully encapsulated rockbolt[END_REF][START_REF] Hyett | A Constitutive Law for Bond Failure of Fully-grouted Cable Bolts Using a Modified Hoek Cell[END_REF] to study the debonding process at the failure interface. Nonetheless, as announced in [START_REF] Hyett | The Effect of Rock Mass Confinement on the Bond Strength of Fully Grouted Cable Bolts[END_REF][START_REF] Kaiser | Bond Strength of Grouted Cable Bolts[END_REF], scatter of results is not negligible because of the short embedment lengths considered. Thus, tests should be carried out two or three times in identical conditions in order to confirm the results. With respect to the grouting material, Lokset SF resin and cementitious grouts (water:cement ratios, w:c=0.35 and w:c=0.40) have been tested. Table 2 compiles the characteristics of all the tests conducted.

After each pull-out test, the samples were cut and the interior was observed. In most cases, bond failure took place at the bolt-grout interface, see photographs 5a and 5b. Debonding at the rock-grout interface was also observed in some cases, especially when the borehole wall was not properly rifled, see figure 5c. Besides, the inspection of the samples has proved that the crater depression that is likely to appear around the mouth hole disrupts the debonding process along the embedment length, particularly when such length is short: as shown in photograph 5d, the failure planes in the grout are parallel to the failure plane in the rock sample.

The analysis of the pull-out tests conducted during the tuning phase of the experimental bench that took place at the beginning of this experimental research has indicated two interesting points: concerning the pull rate (at the beginning, some pull-out tests were performed at a rate of 0.8 mm/s), it has been observed that higher displacement rates privilege shearing off the grouting material between the indentations, especially when the bolt profile is rougher (HA25 rockbolts). The formation of gouge material confirms the considerable degradation at the bolt-grout interface. On the contrary, low pull rates favor a progressive damage of the joint interface. The second point has to do with the crater depression: when this end effect propagates along the embedment length, the test results show important differences with respect to the tests where the embedded length is not disturbed. Figure 6 shows a comparison between two similar tests, except for the breakage of the grout during the pull-out process. As explained before, the altered tests will not be taken into consideration to derive a bond-slip model.

From the laboratory results, it has been concluded that the most influential parameter is undoubtedly the confining pressure: the peak load increases remarkably when the lateral pressure applied increases. The residual phase is also highly dependent on the confinement: when the confining pressure is low, the profile of the bolt is likely to be well reproduced, so that the spacing between the peaks in the residual phase matches the distance between the bolt asperities. For higher confinements, the residual load switches towards a flat decrease. At the same time, as a result of the radial dilation developed (which decreases with lateral pressure), radial tensile cracks appear not only in the grout but also in the rock sample. As expected, the extension of these fractures is more important for lower confinements. Moreover, if the volume of confining oil is maintained constant throughout the test, the increase in pressure can help in the calculation of the sample's dilation. For instance, in the case of FRP rockbolts, the confining pressure increased 1.5 MPa at the peak force when the initial confining pressure was 2 MPa, 0.5 MPa when the initial confining pressure was 5 MPa and only 0.3 MPa when the initial confinement was 10 MPa. As for HA25 rockbolts, the increase in the lateral pressure was less significant: less than 0.5 MPa for an initial confinement of 2 MPa. To further quantify the dilatant effect, pull-out tests have been planned on smooth steel bars.

As a final point, the comparison of two pull-out tests carried out under identical conditions but using two different embedment lengths is shown in figure 8. Several loading-unloading cycles have been executed. The initial slope is approximately 110 kN/mm in both cases and that of the cycles is 150 kN/mm. Even if these results suggest a proportional increase of the peak axial

In situ pull-out test campaign

Laboratory results should, as far as possible, be compared to real case situations where comparable conditions are met.

Within the framework of the ongoing excavation phase at ANDRA's Underground Research Laboratory (URL) located in Bure, 66 pull-out tests were performed in autumn 2009. They were carried out in two technical drifts, GT8 and GAT, oriented in the direction of the maximum horizontal principal stress. Four types of rockbolts were tested: Swellex, CT-Bolts, HA25 and Dywidag 26WR. They were installed in the crosssections between steel arches, with a radial layout. Since HA25 rockbolts have also been tested at laboratory scale, the comparison between laboratory and field results is limited to this type of bar. In the future, Dywidag 26WR continuous thread bars will also be tested in the laboratory and the results will be compared to those obtained in situ.

With regard to the operational conditions, the load was applied using a hollow ram jack and the displacement was measured using a potentiometer. It was decided that the tests would be carried out until either the axial load reached 90% of the yield strength of the bar, T e , or the axial displacement reached 30mm.

Two types of tests were conducted according to the French Standards NF P 94-242-1 and NF P 94-153:

• Constant rate pull-out tests: the pull rate was comprised between 1 and 1.5 mm/min (0.02 mm/s on average); • Creep tests: ten constant load levels (∆F=10%T e ) were applied. The duration of each level increased with the applied load. The former levels lasted 15 minutes and the latter up to 1 hour.

Two grouting materials were used for HA25 rockbolts: Minova Lokset SF resin and a cementitious grout (w:c=0.5). The yield strength of these bars is 245 kN and that of the threaded part is 192 kN; therefore, the applied loads were in general lower than 173 kN. The anchored lengths tested were 2.4 and 3.0 m. Figure 9 shows typical constant rate pull-out tests results. The grouting material and the embedment length are indicated in each case. Four out of the five rockbolts were installed at the bottom of the sidewall; only the resin grouted rockbolt in the drift GT8 was located in the upper sidewall. As a result of the long embedment lengths used, the tests were stopped at very low axial displacements, before any significant shear slip occurred. In some of them, the unload phase was also monitored, but there were no loading-unloading cycles. Besides, it can be inferred from the figure that the resin grouted rockbolts are more rigid than the cement grouted ones. The position of the bolt does not seem to have a major impact on the results. In terms of shear behavior, which was calculated according to [START_REF] Blanco Martín | A New Analytical Solution to the Mechanical Behaviour of Fully Grouted Rockbolts Subjected to Pull-out Tests[END_REF] (since the lengths tested are quite important, it can not be assumed that the shear strength is uniformly distributed), the results for the same pullout tests and their associated uncertainty are shown in figure 10. The solid lines account for the bond-slip models that best reproduce the experimental data and the error bars represent the possible models within an accuracy of ±5 % on the same data. The elastic stiffness of the τ(W) relationship is on average k e =0.60 MPa/mm for the resin grouted bars and k e =0.40 MPa/mm for the cement grouted bars. These values are consistent with previous field tests results [START_REF] Chen | Mechanical behavior of the bamboo-steel composite rock-bolt[END_REF], while a much larger stiffness (about 42.6 MPa/mm) was found in [START_REF] Rong | Testing study on working mechanism of fully grouted bolts of thread steel and smooth steel[END_REF]. Moreover, it seems that the shear strength tends to a perfect plastic behavior characterized by a residual strength of about 1 MPa; nevertheless, in order to derive design information and to fully describe a bond-slip model as explained in [START_REF] Blanco Martín | A New Analytical Solution to the Mechanical Behaviour of Fully Grouted Rockbolts Subjected to Pull-out Tests[END_REF], the axial slips should have been more important so that the debonding front would have been able to propagate all along the interface until the free end of the rockbolt. Anyway, the shear strength of a 0.5 w:c ratio grout can be calculated as stated in [START_REF] Moosavi | Shear strength of Portland cement grout[END_REF] using Eq. ( 6): For the Lokset SF resin, the shear strength is around 25 MPa as reported in [START_REF]Lokset ® Resin Capsules Product Data[END_REF] (laboratory punched shear strength). These values are clearly higher than the maximum stresses reached during the pull-out tests, which means that failure will possibly take place at the interface and not in the materials themselves.

Figure 11 compares the experimental data in figure 9 with the predicted axial load -axial displacement behavior obtained using the new analytical approach and the above bond-slip relationships. As it can be seen, the agreement is quite satisfactory.

TOWARDS A CONSTITUTIVE LAW

From the laboratory results presented in section 4.2, a τ(W) relationship has been derived in each case. Taking into account the short embedment lengths tested, the shear stress has been derived according to Eq. ( 7):

) ( 2 ) ( ) ( W L R W F W - = π τ (7)
Even if the scatter in the results can not be neglected and some tests are less accurate than others, the general trend suggests a bond-slip model composed of an ascending branch followed by an exponential post-peak decay tending to a residual value. The use of two different embedment lengths for a same set of parameters permits to validate the shear strength -shear slip relationship, as it can be seen in figures 12 (FRP rockbolts) and 13 (HA25 rockbolts). Note that, in the blue curve in figure 12, the sudden drops in the shear stress that happen without a major change in the displacement are due to a substantial breakage of the grout during the pull-out test.

The values obtained are the same order of magnitude than those in [START_REF] Moosavi | Bond of cement grouted reinforcing bars under constant radial pressure[END_REF]. The analysis of all tests discloses a rise of the shear stress up to a peak value for a small axial displacement (<6 mm), followed by a steep decay. The higher the confining pressure, the higher the peak strength, but the relationship is not linear. The displacement at which the peak takes place also increases with the confining pressure. It has been observed that the peak shear stress flattens at higher confinements, which is probably due to the decrease of dilation when the interface is sheared off. Concerning the initial linear stiffness, it has been found that it doesn't vary significantly for each type of rockbolt. It is about 14.5 MPa/mm for HA25 bars and 8.8 MPa/mm for FRP rods. The residual shear strength appears for a wide range of axial slips and is largely influenced by the evolution of the test (namely, breakage of the grout or the rock); in fact, the great scatter in the results can be explained by the variability that affects the parameters influencing the bond mechanism. As for the oscillations, their amplitude tends to decrease with the confining pressure. As expected, the wavelength is 16 mm for the steel rockbolts and 10 mm for the FRP bars. With the intention of introducing the effect of the pressure in the constitutive law, the confinement has been varied during some tests while the axial displacement has been kept constant (constant embedment length conditions). So far, the pressure has been changed in the residual phase, i.e. for axial displacements beyond 50 mm. Figure 14 shows the dependence of the residual shear stress on the confining pressure for two resin-grouted steel threaded bars. In the upcoming pull-out tests, the lateral pressure will be also varied at the peak load. Finally, as one could remark, the comparison between laboratory-derived and field-derived τ(W) relationships shows important differences; nevertheless, it is important to note that field and laboratory conditions may differ considerably: the rock sample properties and its degree of damage; the installation of the rockbolt (in situ, the plastic cartridges remain in the borehole, air bubbles are likely to form, etc., whereas in the laboratory the rockbolt is well centered in the borehole, the grouts are carefully prepared before being poured into the holes, …); the confining pressures applied in the laboratory, which may be rather higher than those in field (actually in situ stresses may vary considerably and this is difficult to reproduce at laboratory scale); the pull rate, and the scatter of the results also found in [START_REF] Chen | Mechanical behavior of the bamboo-steel composite rock-bolt[END_REF][START_REF] Rong | Testing study on working mechanism of fully grouted bolts of thread steel and smooth steel[END_REF], among others.

CONCLUSIONS AND PERSPECTIVES

The failure mechanism by debonding of fully grouted rockbolts is reviewed theoretically and experimentally. Concerning the theoretical study, a new analytical solution that predicts the axial load -axial displacement response of a grouted bar is proposed. With reference to the experimental part, a laboratory scale pull-out test campaign is being carried out using a new experimental bench and the results are being analyzed. This campaign aims at the determination of a constitutive law for the rockbolt-grout or rock-grout failure joint interface. For this purpose, the influence of several parameters such as the confining pressure and the profile of the bolt is under study. Special attention is being paid to radial dilation, which develops as a result of the interface geometry and depends heavily on the confining pressure and on the strength of the grout and the rock. Laboratory and field pull-out tests results have been compared and an attempt to explain the differences encountered has been made.

The bond-slip model will be completed by the introduction of dilation and normal pressure and it will be implemented in a FEM code. Furthermore, a similar study will be carried out on classic seven strands and on modified geometry Mini-Cage cable bolts.

Fig. 1 .

 1 Fig. 1. Stress distribution in an elementary length dz.

Fig. 2a .

 2a Fig. 2a. New analytical solution: comparison between a field pull-out test result and the theoretical prediction.

Fig. 2b .

 2b Fig. 2b. New analytical solution: comparison between a laboratory pull-out test result and the theoretical prediction.

Fig. 3 .

 3 Fig. 3. Cutaway section of the new experimental bench.

Fig. 4 .

 4 Fig. 4. Profile of the rockbolts under study.

Fig. 5 .

 5 Fig. 5. Open view of some samples after the pull-out tests.

Fig. 6 .

 6 Fig. 6. Effect of the propagation of the crater effect along the anchored length.

  Figure 7 accounts for the influence of the confining pressure on the axial response. The graphs show two tests conducted at confining pressures of 1.2 and 4.6 MPa respectively (the confinement is the only parameter that changes between the two tests). The bolt profile is almost absent when P=4.6 MPa. The figures below show the aspect of the grout and the rock sample after both tests; as it can be seen, radial fracturing is much more significant when P=1.2 MPa.

Fig. 7 .

 7 Fig. 7. Effect of the confining pressure on pull-out test results. load with the embedment length, further tests are required before any conclusion made.

Fig. 8 .

 8 Fig. 8. Laboratory pull-out tests results. Influence of the embedment length.

Fig. 9 .

 9 Fig. 9. In situ pull-out tests conducted in ANDRA's URL.

Fig. 10 .

 10 Fig. 10. Shear stress (and associated uncertainty) as derived from field pull-out tests.

Fig. 11 .

 11 Fig. 11. New analytical solution: comparison between field experimental data and predicted results.

Fig. 12 .

 12 Fig. 12. Laboratory-derived shear stress -shear slip relationship. FRP rockbolts.

Fig. 13 .

 13 Fig. 13. Laboratory-derived shear stress -shear slip relationship. HA25 rockbolts.

Fig. 14 .

 14 Fig. 14. Effect of the variation of the confining pressure on the shear stress. Residual phase, HA25 rockbolts.

Table 1 .

 1 Mechanical characteristics of the rock sample used

	Property	Value
	Young's modulus, E	25,600 MPa
	Poisson's ratio, ν	0.26
	Density, ρ	2140 kg/m 3
	Uniaxial Compressive Strength, UCS 60 MPa
	Tensile strength, R t	3.1 MPa
	Cohesion, C	12.4 MPa
	Friction angle, φ	46º

Table 2 .

 2 Laboratory pull-out test campaign

	Rockbolt	Confining pressure (MPa)	L(mm)	Grouting material
		15	150	Resin
		10	130	Resin, w:c=0.35
			90	Resin
		5	130	Resin, w:c=0.40
	HA25		90	Resin
		2	130	Resin, w:c=0.35
			90	Resin
		1.2	130 90	Resin Resin
		0	130 90	Resin Resin
		10	130 90	Resin Resin
		5	130	Resin, w:c=0.35
	FRP	2	90 130	Resin w:c=0.35 Resin,
			90	Resin
		0	130 90	Resin Resin

ACKNOWLEDGMENTS

This research is being undertaken in partnership with ANDRA. Rockbolts and grouting materials tested in the laboratory have been supplied by ANDRA, whose Research Department has kindly provided the authors with experimental in situ pull-out tests results. Funding and cooperation from partners within the framework of the European program RFCS (PROSAFECOAL Project) is also fondly acknowledged.


Sylvain Lecomte

Florent Fabienne Desmots

Le Masson

Pascale Le Goff

Denis Michel

Elisabeth S Christians

Yves Le Dréan
email: yves.le-drean@univ-rennes1.fr

F Le Masson

P Le Goff

Dr Yves Le

Roles of Heat Shock Factor 1 and 2 in response to proteasome inhibition: consequence on p53 stability

Keywords: Heat shock factor, MG132, Proteasome, Gankyrin, p53

teaching and research institutions in France or abroad, or from public or private research centers.

Introduction

Vertebrate genome contains 3 to 5 members of the Heat Shock Transcription Factor (HSF) family (for review see [START_REF] Pirkkala | Roles of heat shock transcription factors in regulation of heat shock response and beyond[END_REF][START_REF] Akerfelt | Heat shock factors at a crossroad between stress and development[END_REF]. Within this family, HSF1 and HSF2 possess close related DNA-binding domains but they exhibit some slight differences in Heat Shock Element (HSE) recognition [START_REF] Kroeger | Selection of new HSF1 and HSF2 DNA-binding sites reveals difference in trimer cooperativity[END_REF][START_REF] Yamamoto | Differential recognition of heat shock elements by members of the heat shock transcription factor family[END_REF], suggesting that both factors control different set of genes. HSF1 represents the archetypal stress transcription factor, rapidly activated by a large variety of proteotoxic stimuli. It regulates the stress inducible expression of Heat Shock Proteins (HSPs) but also controls numerous other genes as demonstrated by transcriptome analysis [START_REF] Trinklein | The role of heat shock transcription factor 1 in the genome-wide regulation of the mammalian heat shock response[END_REF]. Recently, it was shown that HSF1, by favouring cellular adaptation and survival in response to environmental stress, also enhances oncogenic transformation [START_REF] Dai | Heat shock factor 1 is a powerful multifaceted modifier of carcinogenesis[END_REF]. In comparison to HSF1, HSF2 is a less efficient transcriptional activator and appears to be differently responsive to stimuli [START_REF] Sistonen | Activation of heat shock factor 2 during hemin-induced differentiation of human erythroleukemia cells[END_REF]. However, inhibition of the proteasome activates both, HSF1 and HSF2 [START_REF] Kawazoe | Proteasome inhibition leads to the activation of all members of the heat-shock-factor family[END_REF]. Proteasome inhibition leads to the accumulation of misfolded proteins and consequently induces expression of all the major HSPs [START_REF] Bush | Proteasome inhibition leads to a heat-shock response, induction of endoplasmic reticulum chaperones, and thermotolerance[END_REF], but Pirkkala and coworkers [START_REF] Pirkkala | Disruption of heat shock factor 1 reveals an essential role in the ubiquitin proteolytic pathway[END_REF], clearly showed that HSF1, but not HSF2, plays a key role in this induction. Nevertheless, to add to the complexity of this proteotoxic response, our group showed that a HSF1/HSF2 heterocomplex was present on the promoter of the chaperone gene Clusterin, following proteasome inhibition [START_REF] Loison | Up-regulation of the clusterin gene after proteotoxic stress: implication of HSF1-HSF2 heterocomplexes[END_REF]. The existence of HSF1/HSF2 heterotrimer was recently confirmed [START_REF] Sandqvist | Heterotrimerization of heat-shock factors 1 and 2 provides a transcriptional switch in response to distinct stimuli[END_REF] and it was demonstrated that HSF2 can act as a modulator of HSF1 activity in response to proteotoxic insults [START_REF] Ostling | Heat shock factor 2 (HSF2) contributes to inducible expression of hsp genes through interplay with HSF1[END_REF]. Hence, the respective role of HSF1 and HSF2 remains unclear when cells are exposed to proteasome inhibitors.

The proteasome/ubiquitin system constitutes the major pathway for regulated degradation of intracellular proteins. The proteasome holoenzyme is a large multi-subunit complex composed of a 20S proteolytic core particle associated with one or two 19S regulatory particles [START_REF] Pickart | Proteasomes and their kin: proteases in the machine age[END_REF]). The 20S core particle is a cylindrical structure made up of four heteromeric rings. The two outer rings are composed of seven different α subunits (named α1 to α7), whereas the two inner rings are composed of seven different β subunits (β1-β7) and formed the catalytic chamber. Although these subunits are evolutionary related and similarly conserved, only β1, β2 and β5 possess proteolytic properties. The 19S regulatory particle comprises approximately eighteen distinct subunits that form a lid controlling the access to the catalytic core particle. Proteins destined to be degraded by proteasome must be conjugated to multi-ubiquitin chain for recognition by the 19S regulatory particle. Dysfunction of the proteasome pathway can lead to many disorders, including cancers and neurodegenerative diseases [START_REF] Paul | Dysfunction of the ubiquitin-proteasome system in multiple disease conditions: therapeutic approaches[END_REF]. Proteasome is crucial for the regulation of cell cycle and apoptosis and its specific inhibition by molecules has emerged as a promising strategy to treat cancers [START_REF] Nencioni | Proteasome inhibitors: antitumor effects and beyond[END_REF]. For example, bortezomib (also known as PS-341 or Velcade®) is the first proteasome inhibitor used in the treatment of multiple myeloma [START_REF] Twombly | First proteasome inhibitor approved for multiple myeloma[END_REF]. In addition, proteasome inhibition may modulate many other transcriptional events [START_REF] Muratani | How the ubiquitin-proteasome system controls transcription[END_REF] and the activity of stress-related transcription factors, such as p53 and HSFs.

To determine the contribution of HSF2 versus HSF1 in cell response to proteasome inhibitors, we used immortalized Mouse Embryonic Fibroblasts (iMEFs) derived from wild type (WT), Hsf1 and/or Hsf2 knock-out (KO) mice [START_REF] Mcmillan | Targeted disruption of heat shock transcription factor 1 abolishes thermotolerance and protection against heat-inducible apoptosis[END_REF][START_REF] Mcmillan | Heat shock transcription factor 2 is not essential for embryonic development, fertility, or adult cognitive and psychomotor function in mice[END_REF]. We found that both factors are essential for cell viability but have distinct actions: as previously shown, HSF1 is required in the induction of pro-survival HSP expression and in contrast, HSF2 is involved in the regulation of proteasome subunit expression.

Results

Proteasome inhibition is severely toxic for HSF1 and/or HSF2-deficient cells.

To determine the specific contribution of HSF1 and HSF2 to the cellular response to proteasome inhibition, we first examined the cytotoxic effect of two proteasome inhibitors: the peptide aldehyde MG132, a reversible inhibitor and the peptide epoxyketone Epoxomicin, an irreversible inhibitor. To assess viability of iMEFs cells, we utilized the quantitative colorimetric MTT test as cytotoxicity assay and we established concentration-effect curves after a 16 h treatment. With high concentrations of MG132 (830 nM) or Epoxomicin (250 nM) Hsf1 -/-, Hsf2 -/-or Hsf1 -/-& Hsf2 -/-iMEFs were significantly more sensitive to these proteasome inhibitors than WT cells (Student's t-test, p < 0.05 and p<0.001 respectively) (Fig. 1A and 1B,respectively). By comparison, we treated cells with Thapsigargin that induces a proteotoxic stress specific to the reticulum endoplasmic without affecting proteasome activity and without activating HSF. In contrast to proteasome inhibition, Thapsigargin treatment had the same effect on the four cell lines. These data clearly showed that the higher sensitivity of HSF1-and HSF2-deficient cells was specific to proteasome switch off and was not a direct consequence of a general alteration of stress response in these KO cells (Fig. 1 C).

To evaluate whether the decline of MTT values after proteasome inhibition was due to an antiproliferative or a cytotoxic effect, we conducted a flow cytometric analysis of cellular viability. The WT or HSF-deficient iMEFs were treated for 16 h with 1 µM of MG132, and then apoptotic levels were determined by staining with annexin V-fluorescein isothiocyanate (FITC) and 7-aminoactinomycin D (7-AAD) (Fig. 1 D and supplemental Fig. S1). Mortality of all the cell lines was increased in MG132 treated samples, and apoptosis level in HSFdeficient cells was found 2 fold higher compared to WT cells. These data revealed that both factors, HSF1 and HSF2, are critical for cell survival when proteasome is inhibited.

HSP induction is differently affected in Hsf1 -/-and Hsf2 -/-iMEFs HSPs are important chaperones induced by proteotoxic stress where they contribute to cell death resistance (Beer HM, 2005). Thus we analysed the inducible expression of two HSPs: HSP70 and HSP25. Messenger RNA levels of Hsp25 and Hsp70 in WT, HSF1 and/or HSF2-deficient iMEFs were compared in absence or presence of MG132 (Fig. 2A and2B).

After MG132 exposure, only iMEFs expressing HSF1 (either WT or Hsf2 -/-) were able to exhibit a strong induced expression of both chaperones, whereas iMEFs deficient in HSF1 (Hsf1 -/-and Hsf1 -/-& Hsf2 -/-) did not. Those data indicated that expression of inducible Hsp70 and Hsp25 is highly dependent on the presence of HSF1. These results were confirmed by immunoblot (Fig. 2C), HSP25 and HSP70 are strongly increased in WT cells but not in HSF1-depleted cells (Hsf1 -/-and Hsf1 -/-& Hsf2 -/-). Moreover, HSF2-depleted cells display higher levels of HSP25 and HSP70 than those observed in WT iMEFs -wich could be explained by a lower level of protein degradation and thus an increase in protein's half-life in those Hsf2 -/-cell lines. Hence, it was unlikely that the high sensitivity of Hsf2 -/-cells to proteasome inhibition could be explained by the lack of HSPs. This suggested that another mechanism should be involved.

Proteasome activity is decreased in Hsf2 -/-iMEFs

To test this possibility that proteasome function would be natively affected, an in vitro assay based on the measurement of the chymotrypsin-like activity of proteasome present in crude protein extract was first used. Secondly, the proteasome activity at the cellular level was evaluated, by setting up an assay based on the degradation of a proteasome-targeted protein.

A fluorescent proteasome substrate (Z-LLVY-AMC) was incubated with cellular extracts from the different iMEFs cell lines. This peptide was hydrolyzed by chymotrypsinlike activity of proteasome releasing the fluorophore that could be assessed with a spectrofluorimeter (Fig. 3A). Hsf2 -/-and Hsf1 -/-& Hsf2 -/-cellular extracts contain a lower amount of active proteasome (0.36 +/-0.11 and 0.21 +/-0.04, respectively) compared to WT iMEFs (0.96 +/-0.20). Decrease of proteasome activity measured in Hsf1 -/-cells (0.63 +/-0.14) was less striking but still significant. To ascertain that measured fluorescence was due to proteasome activity, crude extracts were incubated with the peptide and 10 µM of MG132 and no fluorescence could be measured, indicating that this inhibitor clearly abolished the previously assessed activity.

To confirm in vivo this observation at the cellular level, expression vector coding for the fusion protein uncleavable Ubiquitin-Green Fluorescent Protein (Ubi-GFP) was transfected in iMEFs. The mutated non cleavable ubiquitin moiety within the fusion protein serves as template to poly-ubiquitination, which is addressed to proteasome for degradation.

Thus, intracellular accumulation of the unstable Ubi-GFP requires partial or total inhibition of proteasome/ubiquitin system. Ubi-GFP expression vector was co-transfected with a plasmid coding for EGFP, as internal control to evaluate the difference of transfection efficiencies between the iMEFs cell lines. Then, the protein levels of GFP were probed by immunoblotting, using the same antibody (Fig. 3B). In all cell lines (except the double knockout iMEFs), the EGFP control is highly expressed, demonstrating that the transfections were equally efficient. We found that in WT and Hsf1 -/-cells, the Ubi-GFP specific bands were not visible in untreated cells while it became slightly detectable when cells were incubated with 1 µM of MG132. These data showed that Ubi-GFP was efficiently targeted to proteasomal degradation and demonstrated that exposure to 1 µM MG132 could not block totally the intracellular proteasome activity. By contrast, Ubi-GFP was significantly more stable in cells deficient in HSF2 (Hsf2 -/-and Hsf1 -/-& Hsf2 -/-), especially after MG132 treatment (Fig. 3B, lanes 6 and 8, respectively). This indicated that HSF2-deficient cells exhibited a reduced proteasome activity either due to functional defect or reduced amount of the proteasome machinery in comparison to WT.

Partial but significant depletion of Ubiquitin-proteasome machinery in Hsf2 -/-iMEFs

To determine whether the decrease of proteasome activity was linked to reduced level of proteasome subunit expression, 10 subunits were selected (5 located in the 20S particle and 5 located in the 19S particle -Table 1) based on the work of Kruger's team about proteasome expression in response to proteasome inhibition [START_REF] Meiners | Inhibition of proteasome activity induces concerted expression of proteasome genes and de novo formation of Mammalian proteasomes[END_REF] and the level of transcripts was analyzed by real time PCR analysis. The expression level of two ubiquitin genes (Ubb and Ubc) was also analyzed. As summarized in Table 1, a decreased expression was observed in absence of HSF1 for some proteasome subunits and for Ubb and Ubc but at a lower level than observed in HSF2-deficient cells. In fact, the level of the majority of the 20S and 19S subunits likewise the two ubiquitin genes decreased by a factor 2 or 3 fold in HSF2deficient cells. However, mRNA levels of two subunits, Psmb4 and Psmc5 were unchanged in HSF-deleted cells compared to WT, suggesting that all constitutive subunits are not regulated in the same way. In addition, it is interesting to note that the level of the β5 catalytic subunit (Psmb5) was significantly lower in Hsf2 -/-and Hsf1 -/-& Hsf2 -/-cells (Table 1 and Fig. 4A), which is consistent with the decrease of proteasome activity measured in these cells (Fig. 3). To ascertain that this decrease of expression was due to the lack of HSF2, we stably transfected Hsf2 -/-iMEFs with a plasmid coding for HSF2 or a control vector (Fig. 4C and supplemental Fig. S2). Reintroduction of HSF2 allowed a rescue of Psmb5 expression which was 2-3 fold higher in transfected cells with Hsf2 vector in comparison to empty vector.

Increased stability of p53 in HSF2-depleted cells

As shown in Fig. 4B, Psmd10 or gankyrin, an oncogene overexpressed in human hepatic cancers [START_REF] Higashitsuji | Reduced stability of retinoblastoma protein by gankyrin, an oncogenic ankyrin-repeat protein overexpressed in hepatomas[END_REF] was 2-4 fold down-regulated in HSF2-deficient iMEFs and its expression was totally rescued when cells were transfected with Hsf2 vector (Fig. 4D). As gankyrin is also known to interact with the MDM2 ubiquitin ligase and to play a pivotal role in controlling the level of p53 by proteasomal degradation [START_REF] Higashitsuji | The oncoprotein gankyrin binds to MDM2/HDM2, enhancing ubiquitylation and degradation of p53[END_REF], we wanted to determine whether the lower level of gankyrin affects intracellular p53 concentration. Using untreated cells, in immunoblot assay, endogenous p53 was hardly visible in WT and Hsf1 -/-iMEFs, whereas it was dramatically increased in both Hsf2 -/-and in Hsf1 -/- & Hsf2 -/-iMEFs (Fig. 5A). Real time PCR analysis revealed that there was no significant increased level of p53 transcripts in cell lines which exhibited higher p53 protein content, suggesting that this p53 up-regulation in HSF2-deficient cells was due to protein stabilization (Fig. 5B). Finally, a pulse-chase experiment using p53 antibody was performed to assess p53 half-life in our model. As shown in Fig. 5C, endogenous p53 in WT iMEFs exhibited a very short half-life (less than 1 h). In Hsf1 -/-iMEFs p53 half-life was slightly longer than in WT cells as previously described [START_REF] Jin | Heat shock factor 1 deficiency via its downstream target gene alphaB-crystallin (Hspb5) impairs p53 degradation[END_REF]. P53 was yet detected after 2 hours of chase. This result was in accordance with the decrease of proteasome activity, observed in HSF1deleted cells. In sharp contrast with this modest increased stability, p53 was dramatically stabilized to a half-life of more than 9 h, in Hsf2 -/-cells. To confirm this stabilization, p53 immunoblotting was performed in primary MEF from WT, Hsf1 -/-and/or Hsf2 -/-mice. In those cells, the basal endogenous p53 was not detectable (Fig. 5D). In contrast, when cells were treated with 1 µM of MG132, p53 was slightly increased in WT and Hsf1 -/-MEFs whereas a strong accumulation was detectable in Hsf2 -/-and Hsf1 -/-& Hsf2 -/-MEFs. All together our data suggest that proteasome and gankyrin down-regulations in Hsf2 -/-cells are accompanied by stabilization of the short-lived protein p53.

Discussion

Proteasome inhibition causes an accumulation of altered proteins, generating a proteotoxic stress, which was shown to simultaneously activate both HSF1 and HSF2 [START_REF] Kawazoe | Proteasome inhibition leads to the activation of all members of the heat-shock-factor family[END_REF]. Nevertheless the respective roles of HSF1 and HSF2 remained unclear. We took advantage of genetically modified cells to further clarify the specific function of each factor in the cellular response to such a stress. We found that HSF1 and HSF2 were both critical for the resistance to proteasome inhibition. However, HSF1 and HSF2 are involved in different mechanisms.

HSF1 deficiency dramatically prevented the induced expression of Hsp25 and Hsp70 after proteotoxic stress. This was in agreement with previous works showing that HSF1 is the major factor involved in proteotoxic response and that it could not be compensated by HSF2 alone [START_REF] Pirkkala | Disruption of heat shock factor 1 reveals an essential role in the ubiquitin proteolytic pathway[END_REF]. As HSP25 and HSP70 promote cellular survival by preventing protein aggregation, facilitating refolding and directly inhibiting apoptotic signalling [START_REF] Beere | Heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome[END_REF][START_REF] Bruey | Hsp27 negatively regulates cell death by interacting with cytochrome c[END_REF], Hsf1 -/-cells lacked an important protective mechanism which could explain the observed reduction in cell survival. In contrast to Hsf1 deletion, Hsf2 deletion did not prevent induction of Hsp25 and Hsp70 expression. Thus, the high expression of protective HSP in Hsf2 -/-iMEFs led us to conclude that HSF2 and cell sensitivity to proteasome inhibition were linked by a different mechanism. According to this hypothesis, we were interested to proteasome subunit expression and found that it was particularly affected in

Hsf2 -/-cells.
Little is known about the regulation of basal expression of mammalian proteasome subunits. Recently, one study suggested that each type of proteasome subunits could be regulated by common unknown transcription factors [START_REF] Sato | Proteasome subunits are regulated and expressed in comparable concentrations as a functional cluster[END_REF]. It was found that inactivation of HSF1 does not alter proteasome expression [START_REF] Taylor | Proteasome activity or expression is not altered by activation of the heat shock transcription factor Hsf1 in cultured fibroblasts or myoblasts[END_REF] and our data are in accordance with this report as we show that Hsf1 -/-iMEFs exhibit a slight but not significant decrease of proteasome subunit expression. However, this slight decrease could be explained by interdependence between HSF2 and HSF1 [START_REF] Loison | Up-regulation of the clusterin gene after proteotoxic stress: implication of HSF1-HSF2 heterocomplexes[END_REF][START_REF] Sandqvist | Heterotrimerization of heat-shock factors 1 and 2 provides a transcriptional switch in response to distinct stimuli[END_REF]. Thus, one could hypothesize that the lack of HSF1 would affect HSF2 binding and consequently reduce HSF2 activity.

Currently, we do not know if HSF2 regulates proteasome subunit expression indirectly or by direct binding to the promoters of the corresponding genes. By in silico analysis we searched for the presence of HSE, within genes encoding proteasome subunits and found that the majority of those genes possess putative degenerated HSEs. For example, the gankyrin gene contains a conserved HSE into its first intron. This sequence was able to bind HSF in electrophoretic mobility shift assay, but we hardly detected in vivo HSF2 binding by chromatin immunoprecipitation assay (data not shown). The difficulty of detecting HSF2 bound to gankyrin HSE could be explained by the fact that this interaction could be very transient. Recently, HSF2 was found to ephemerally bind to Hsp promoters during mitosis and that interaction favoured the basal expression of Hsps. [START_REF] Xing | Mechanism of hsp70i gene bookmarking[END_REF][START_REF] Wilkerson | HSF2 binds to the Hsp90, Hsp27, and c-Fos promoters constitutively and modulates their expression[END_REF]. So, it is possible that the role of HSF2 in bookmarking that maintains an open chromatin at the promoters of certain genes entails the constitutive expression level of proteasome subunits. To test this hypothesis, Formaldehyde Assisted Isolation of Regulated Element assay was performed (supplemental Fig. S3) and showed that chromatin was more compacted at gankyrin transcription start site in HSF-deficient cells than in WT cells. However, another mechanism described in yeast could be implicated. An indirect transcriptional network involving HSF and RPN4 was demonstrated [START_REF] Hahn | A stress regulatory network for co-ordinated activation of proteasome expression mediated by yeast heat shock transcription factor[END_REF]. This latter protein corresponds to the main transcription factor implied in the expression of yeast proteasome subunits but no mammalian homolog has been described to date.

The decreased expression of proteasome subunits has consequence for cell physiology.

Alteration of proteasome activity has been implicated in the pathogenesis of cancers [START_REF] Schwartz | The ubiquitin-proteasome pathway and pathogenesis of human diseases[END_REF] and in some cells, a clear relationship has been established between increased expression of proteasome and proliferation or resistance to apoptosis [START_REF] Fuchs | Increased expression and altered subunit composition of proteasomes induced by continuous proteasome inhibition establish apoptosis resistance and hyperproliferation of Burkitt lymphoma cells[END_REF]. We found that decrease of catalytic subunit β5 and gankyrin in HSF2depleted cells was accompanied by p53 stabilization. It would be interesting to determine whether re-introduction of gankyrin in Hsf2 -/-iMEF could restore the level of p53, knowing that it was already demonstrated that gankyrin overexpression accelerates the degradation of p53 in various cell models, including MEF [START_REF] Higashitsuji | The oncoprotein gankyrin binds to MDM2/HDM2, enhancing ubiquitylation and degradation of p53[END_REF]. Mivechi's group described a mechanism linking HSF1-deficiency and p53 stabilization [START_REF] Homma | Demyelination, astrogliosis and accumulation of ubiquitinated proteins, hallmarks of CNS disease in hsf1-deficient mice[END_REF][START_REF] Jin | Heat shock factor 1 deficiency via its downstream target gene alphaB-crystallin (Hspb5) impairs p53 degradation[END_REF]. Our study confirms a role for HSF1 in the regulation of p53 with a slight stabilization of this protein in pulse chase experiment compared to WT cells. However, our work completes the mechanism by introducing HSF2 as a new actor. The iMEFs used in this study have been immortalized with T antigen of SV40 virus that is known to bind to p53 and blocks its transcriptional activity [START_REF] Pipas | Role of T antigen interactions with p53 in tumorigenesis[END_REF] which could permit its stabilization in our model. However our results (p53 stabilization) were confirmed by p53 immunoblot in primary MEFs. Furthermore, it was found that SV40 transformation does not affect the protein control machinery [START_REF] Cantalupo | Cell-type specific regulation of gene expression by simian virus 40 T antigens[END_REF], which emphasizes that the decrease of ubiquitin-proteasome system expression is related to the loss of Hsf2 gene confirmed by HSF2 rescue.

Development of inhibitors of both HSF1 and HSF2 could be a valuable approach to increase effectiveness of cancer therapy. By blocking HSF1, it is possible to alter the adaptive transcriptional response that allows the expression of anti-apoptotic chaperones [START_REF] Zaarur | Targeting heat shock response to sensitize cancer cells to proteasome and Hsp90 inhibitors[END_REF]. Therapeutic use of proteasome inhibitors remains limited because of the selection of resistant cells. It was found that bortezomib-adapted cells showed increased expression of β5 and β2 proteasome subunits [START_REF] Oerlemans | Molecular basis of bortezomib resistance: proteasome subunit beta5 (PSMB5) gene mutation and overexpression of PSMB5 protein[END_REF][START_REF] Rückrich | Characterization of the ubiquitin-proteasome system in bortezomib-adapted cells[END_REF], and we found that one of these subunits was down-expressed in absence of HSF2. Moreover, by blocking HSF2, the level of intracellular proteasome is expected to decrease and potentially to increase the sensitivity to proteasome inhibition, even in resistant clones. Recently, a close correlation was demonstrated between expression levels of proteasome subunits, their enzymatic activity and the sensitivity of cells to proteasome inhibitor [START_REF] Busse | Sensitivity of tumor cells to proteasome inhibitors is associated with expression levels and composition of proteasome subunits[END_REF]. Finally, a decrease of gankyrin level in Hsf2-blocked cells would permit overexpression of p53 that will in turn participate in the apoptosis of treated cells.

In conclusion, our work in iMEFs brought new insights in the respective roles of HSF1 and HSF2, the most abundantly expressed HSF in organisms. Beyond these fundamental interests in a better understanding of transcription factor, the data presented here confirm that HSF1 and now HSF2 could be targeted with benefits in cancer therapy.

Materials and methods

Cell culture

Mouse Embryonic Fibroblasts Wild Type (WT), deleted for HSF1 (Hsf1 -/-), HSF2 (Hsf2 -/-) or both genes (Hsf1 -/-& Hsf2 -/-) were isolated from KO mice [START_REF] Mcmillan | Targeted disruption of heat shock transcription factor 1 abolishes thermotolerance and protection against heat-inducible apoptosis[END_REF][START_REF] Mcmillan | Heat shock transcription factor 2 is not essential for embryonic development, fertility, or adult cognitive and psychomotor function in mice[END_REF] and were immortalized using SV40 T antigen by Dr V. Mezger (UMR CNRS 7216, Paris, France). Cells were maintained in DMEM 4500 mg/L glucose (Gibco, CA, USA) supplemented with 10 % Fetal Bovine Serum (Biowest, France), 4 mM L-glutamine, Non Essential Amino-Acids and 100 Units/mL penicillin, 100 mg/mL streptomycin (Gibco, CA, USA). All cells were grown at 37°C under 5 % CO 2 .

For rescue experiments Hsf2 -/-iMEFs were stably transfected with plasmid coding for Hsf2α and HSF2 expression was confirm by RT-PCR and HSF2 immunoblotting in each selected clone (supplemental Fig. S2).

Plasmid constructs and transfection

An ubiquitin tagged green fluorescent protein (GFP) was previously used to quantify ubiquitin-proteasome dependent proteolysis in living cells [START_REF] Dantuma | Short-lived green fluorescent proteins for quantifying ubiquitin/proteasome-dependent proteolysis in living cells[END_REF]. For this purpose, the DNA fragment from the pDG268 plasmid (kindly provided by Dr D. Gray, university of Ottawa, Canada), encoding an ubiquitin-EGFP fusion protein [START_REF] Tsirigotis | Analysis of ubiquitination in vivo using a transgenic mouse model[END_REF], was transferred into pCDNA3 expression vector (Invitrogen Life science, CA, USA).

Then, the carboxy-terminal glycine in ubiquitin was converted into a valine using the site directed mutagenesis kit QuickChange (Stratagene, CA, USA), to block cleavage of the ubiquitin moiety by cellular de-ubiquitinases and thus render the entire fusion prone to degradation by the ubiquitin-proteasome system. The vector coding for non cleavable ubiquitin fused to GFP (Ubi-GFP) was co-transfected with a plasmid coding for EGFP (pEGFP-C3, Clontech, CA, USA), as internal control of transfection efficiency. All transfections were performed with JetPEI (Polyplus Transfection, France), according to manufacturer's instructions. Cells were harvested after 24 h of transient expression and 7 h of treatment with DMSO or 1 µM of MG132 (Biomol, PA, USA). Relative quantity of these two GFP proteins was assessed by immunoblotting using anti-GFP antibodies (Invitrogen Life Science, CA, USA).

Cell toxicity assay (MTT assay)

WT, Hsf1 -/-, Hsf2 -/-and Hsf1 -/-& Hsf2 -/-iMEFs were spread in 96 wells-plate and were treated with increasing doses of proteasome inhibitors Epoxomicin (Sigma Aldrich, MO, USA), MG132 (Biomol, PA, USA), or with Thapsigargin (Sigma Aldrich, MO, USA), an inductor of endoplasmic reticulum stress. Cells were treated at the indicated concentration for 16 h.

Following treatment, MTT [1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan] (Sigma Aldrich, MO, USA) was added to cells at final concentration of 0.5 mg/ml. Cells were incubated for 3 h at 37°C under 5 % CO 2 . The medium was then discarded and formazan crystals were dissolved in 100 µl of isopropanol with 0.1 % SDS and 0.05 N HCl for 1 h at obscurity. Absorbance was read at 570 nm and results were expressed as percentage of untreated cells.

Real Time PCR analysis

One million cells for each iMEFs lines was plated into 10 cm plate and harvested after 24 h of culture. Total RNAs were extracted with Trizol reagent (Invitrogen Life Science, CA USA) according to the manufacturer's procedure. Then 5 µg of RNA were retrotranscribed using M-MLV RT (Invitrogen Life Science, CA USA). Real time PCR was performed using 12.5 ng of cDNA, 300 nM of primers (referred to supplemental data) and 1X SybrGreen, on MiniOpticon Real Time PCR (Bio-Rad, CA, USA). Amplification of each studied gene was normalized using amplification of three housekeeping genes Gapdh (Glyceraldehyde-3phosphate dehydrogenase), Hprt1 (Hypoxanthine guanine phosphoribosyl transferase 1) and Tbp (TATA box binding protein). Data were analysed using qBase software [START_REF] Hellemans | qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data[END_REF].

Measurement of proteasome activity

Proteasome activity was measured in accordance with the protocol described by Kisselev and Goldberg [START_REF] Kisselev | Monitoring activity and inhibition of 26S proteasomes with fluorogenic peptide substrates[END_REF]. Briefly, cells were harvested in a lysis buffer that maintained 26S proteasome integrity. After incubation on ice during 45 min, the cytosols were squeezed out by centrifugation at 14000 rpm at 4°C for 15 min. 15 µg of proteins were loaded on opaque white 96 well-plates. Volume was adjusted at 90 µl with 26S proteasome assay buffer. Thereafter, 10 µl of Z-LLVY-AMC (Calbiochem, England) at 1 mM stock solution was added and plate was incubated at 37°C. Fluorescence was measured after 15 min and 30 min using a fluorimeter (Dynatech Laboratories, VA, USA) with excitation at 365 nm wavelength and emission at 450 nm wavelength.

Protein extracts and immunoblot analyses

Cells extracts were prepared using osmotic shock methods like previously described [START_REF] Loison | Up-regulation of the clusterin gene after proteotoxic stress: implication of HSF1-HSF2 heterocomplexes[END_REF]. Protein extracts were separated on polyacrylamide gel and transferred to nitrocellulose membrane (Amersham Bioscience, England). Anti-Hsp25 (sc-1084), anti-Hsp70 (SPA-810), anti-β-actin (A-1978) antibodies were purchased from Santa Cruz Biotechnology (CA, USA), Stressgen (MI, USA) and Sigma Aldrich (MO, USA) respectively. Anti-p53 antibody was a gift from Dr T. Soussi, Université Pierre et Marie Curie, Paris, France.

Pulse-Chase Assays

Wild type, Hsf1 -/-and Hsf2 -/-iMEFs were washed with PBS and preincubated for 30 min with methionine-and leucine-free DMEM, containing 10 % dialyzed Fetal Bovine Serum and 4 mM glutamine. Cells were labeled for 1 h 30 in the same medium with 10 µCi/ml of L-[ 35 S]methionine (PerkinElmer, France), and chased in complete media. At various times after termination of labeling, cells were isolated in PBS 0.5% Triton X-100 supplemented by protease inhibitors (Roche Applied Science, France) and incubated 30 min on ice. Proteins were immunoprecipitated overnight at 4°C with 5 µg of monoclonal anti-p53 antibody in PBS supplemented with NaCl 600nM, Triton X-100 4% and protease inhibitors (Roche Applied Science, France). Immune complexes recovered by protein-G Sepharose were washed three times with PBS supplemented with Triton X-100 1% and NaCl 150 mM. Immune complexes were electrophoretically separated on SDS-PAGE gels. Gels were fixed and dried and subjected to autoradiography. Corresponding lysates (lysates 35 S) were loaded as control for equal amount of proteins, as well as equal incorporation of 35 S. (D) Primary MEFs from WT, Hsf1 -/-and/or Hsf2 -/-animals were treated with DMSO (cont) or with 1 µM of MG132 for 16 h. Protein extracts were prepared and p53 western blotting was realized. β-actin was used as loading control.

Supplemental data

Flow cytometry analysis

WT, Hsf1 -/-, Hsf2 -/-and Hsf1 -/-& Hsf2 -/-iMEFs were plated in 6 well-plate and treated with 1 µM MG132 for 16 h.

Formaldehyde Assisted Isolation of Regulatory Elements (FAIRE)

WT, Hsf1 -/-and Hsf2 -/-iMEFs were grown in 15 cm plates to 60-70% confluence. Then cells were cross-linked with 1% formaldehyde for 10 min at room temperature. Formaldehyde was poured off and cells were incubated with 125 mM glycine for 5 min at room temperature.

iMEFs were rinsed with ice cold PBS and harvested. Cells were lysed with 1% SDS, 10mM

EDTA and 50mM Tris-HCl pH 8.1 supplemented with protease inhibitor cocktail (Roche Applied Science, France) and sonicated for 14 min (30 s on / 30 s off cycles) using a Bioruptor (Diagenode) set up at the highest intensity. 20 µl of chromatin were taken as input.

FiguresFig. 1 :

 1 Figures

Fig. 2 :

 2 Fig. 2: HSF dependent response of Hsp25 and Hsp70 to proteasome inhibition. WT, Hsf1 -/-, Hsf2 -/-and Hsf1 -/-& Hsf2 -/-iMEFs were treated with DMSO (cont) or with 1 µM of MG132 for 10 h. Hsp70 (A) and Hsp25 (B) transcript levels were measured by real time PCR. Expression data were normalized by reference genes (see materials and methods) and analyzed with qBase software. Results were expressed in fold induction, normalized to DMSO sample of each cell line. Data are the means of three independent experiments +/-SD.(C) Corresponding immunoblot analysis of HSP70 and HSP25 expression in response to 1 µM of MG132 for 10h in WT, Hsf1 -/-, Hsf2 -/-and Hsf1 -/-& Hsf2 -/-iMEFs. β-actin served as loading control.

Fig. 3 :

 3 Fig. 3: Proteasome catalytic activity in Hsf-deficient cells. (A) In vitro assay using 15 µg of protein extracts from WT, Hsf1 -/-, Hsf2 -/-and Hsf1 -/-& Hsf2 -/-iMEFs incubated with 100 µM of peptide coupled to a fluorophore (Z-LLVY-AMC) in presence of DMSO (cont) or with 10 µM of MG132. The fluorescence was read after peptide cleavage by the proteasome's chymotrypsin-like activity. The relative catalytic activity was expressed in fluorescence (Arbitrary Unit) per minute per microgram of proteins (Fluo/min/µg of prot). Data are the means for three independent experiments +/-SD (Student's Test compare to WT cells. * p<0.05, *** p<0.001). (B). In vivo assay: plasmid coding for the fusion protein Ubiquitin-GFP (Ubi-GFP) was co-transfected with a plasmid coding for EGFP used as transfection efficiency control. Ubiquitin part of the fusion protein serves as template for ubiquitination, rending the protein labile. Cells were treated with DMSO (Cont) or with 1 µM of MG132 for 7 h and stabilization of Ubi-GFP protein was assessed by anti-GFP immunoblot.

Fig. 4 :

 4 Fig. 4: Transcript levels of proteasome subunits in HSF-deficient iMEFs and HSF2 rescued iMEFs. WT or HSF-deficient iMEFs (Hsf1 -/-. Hsf2 -/-and Hsf1 -/-& Hsf2 -/-) were plated and total RNA were extracted the day after. Relative basal expression of two proteasomal subunits Psmb5 (A) and Psmd10 (B) were assessed by real time PCR and normalized using three reference genes Gapdh, Hprt1 and Tbp. Transcript levels in HSFdeficient cells were presented as relative value to WT. Data are the means for four independent experiments +/-SEM. (Student's test compared to WT cells. * p<0.05, ** p<0.01, *** p<0.001). For rescue experiments cells were transfected with PCR3.1 as control vector (white histogram) or PCR3.1 expressing HSF2 (black histogram). Three independent stable clones of each transfection were selected (cl 1, 2, 3 for control vector transfection and cl 4, 5, 6 for HSF2 transfection). Psmb5 (C) and Psmd10 (D) expression level were measured by real-time PCR in each clone and in WT cells (grey histogram). Transcript levels in each stable clone and in WT cells were presented as relative value to PCR3.1 cl 1 for three independent experiments +/-SEM. Data were analyzed with qBase software.

Fig. 5 :

 5 Fig. 5: Stabilization of p53 in HSF2-deficient cells. (A) Untreated immortalized Mouse Embryonic Fibroblast (iMEFs) derived from WT or knock-out Hsf1 and/or Hsf2 mice (Hsf1 -/-, Hsf2 -/-and Hsf1 -/-& Hsf2 -/-) were analyzed by Western immunoblotting using antibodies against p53 and β-actin as control. (B) p53 gene expression was compared in WT, Hsf1 -/-, Hsf2 -/-and Hsf1 -/-& Hsf2 -/-iMEFs, by real time RT-PCR. Data were normalized by three reference genes Gapdh, Hprt1 and Tbp and analyzed using qBase software. Histograms represent relative expression means +/-SD of four independent experiments. (C) WT, Hsf1 -/- and Hsf2 -/-iMEFs were grown and metabolically labeled with L-[ 35 S] methionine for pulsechase study. Labeled proteins were immunoprecipitated using anti-p53 antibody and subjected to denaturing electrophoresis. Lysate 35 S was used as loading and 35 S incorporation control.

  Cells were trypsined and labeled with Annexin-V FITC (Becton Dickinson Pharmigen, CA, USA) and 1 µg of 7-Aminoactinomycin D (7-AAD) (Sigma Aldrich, MO, USA) for 15 min at room temperature and at obscurity. Apoptosis was performed with FACScalibur flow cytometer (Becton Dickinson, NJ, USA) by analyzing 10 000 events. Data analyses were performed using Cell Quest Pro software (Becton Dickinson, NJ, USA).

Fig. S1 :

 S1 Fig. S1: Representative flow cytometry analysis of cellular apoptosis after MG132 exposure. WT, Hsf1 -/-, Hsf2 -/-and Hsf1 -/-& Hsf2 -/-iMEF were treated with DMSO (left panel) or with 1 µM of MG132 for 16 h (right panel). Then apoptosis level was assessed by flow cytometry using annexin-V fluorescein isothiocyanate (FITC) and 7-aminoactinomycin D (7-AAD) labeling. A typical log fluorescence intensity of annexin V-FITC and 7-AAD plot with quadrant delimiting regions with distinct sub-populations of events is shown. Percentages of cells present in each phase were analyzed using Cell Quest Pro software and indicated on graph.

  The soluble extracts were subjected to three consecutive phenol-chloroform isoamylic acid extractions to expand in open chromatin. To reverse cross-linking samples were incubated at 65 °C overnight. Finally DNA was purified using Nucleospin Extract II (Macherey Nagel, Germany). Data were analyzed by real time PCR with 0.5 µl of inputs and 3 µl of chromatin subjected to phenol-chloroform extractions.

Fig. S3 :

 S3 Fig. S3: FAIRE-Real Time PCR analysis of chromatin at the transcription start site of Psmd10 and on clusterin HSE in WT, Hsf1 -/-and Hsf2 -/-iMEFs. Compaction of chromatin at the transcription start site of Psmd10 and one clusterin HSE was abritary fixed to 1 in WT cells and data obtained from HSF-deficient cells were expressed in relative FAIRE enrichment to WT cells. Mean for three experiments +/-SD (* p<0.05 Student's t Test). Data obtained clearly showed that chromatin was more compacted at the transcription start site of Psmd10 in HSF-deficient cells than WT iMEFs.

Table 1 : Transcript levels of proteasome subunits and ubiquitin in iMEFs WT and HSF- deficient.

 1 RNA level of five subunits of 20S particle, five subunits of the 19S particle and Ubiquitin B and C were measured by real time PCR and normalized using three reference genes Gapdh, Hprt1 and Tbp. Results are expressed as relative value to WT. Data are the mean for four independent experiments +/-SD.

		Proteasome subunit	iMEF WT	+/-SD	iMEF Hsf1-/-	+/-SD	iMEF Hsf2-/-	+/-SD	iMEF Hsf1-/-& Hsf2-/-	+/-SD
		Psma1	1,00	0,603	0,68	0,409	0,38	0,113	0,25	0,077
	Particle 20S	Psma5 Psmb2 Psmb4	1,00 1,00 1,00	0,909 0,408 0,443	0,98 0,69 1,31	0,948 0,252 0,303	0,57 0,46 0,78	0,503 0,189 0,164	0,72 0,38 0,98	0,518 0,179 0,237
		Psmb5	1,00	0,32	1,25	0,43	0,50	0,17	0,27	0,08
		Psmc4	1,00	0,377	0,95	0,727	0,41	0,151	0,23	0,086
	Particle 19S	Psmc5 Psmd3 Psmd4	1,00 1,00 1,00	0,354 0,586 0,328	1,09 1,00 1,03	0,214 0,515 0,243	1,21 0,66 0,79	0,256 0,273 0,238	1,28 0,48 0,82	0,329 0,294 0,273
		Psmd10	1,00	0,252	0,51	0,287	0,31	0,058	0,20	0,052
	Ubiquitin	Ubb Ubc	1,00 1,00	0,352 0,658	0,66 0,37	0,211 0,138	0,35 0,23	0,171 0,113	0,30 0,14	0,124 0,066

Table primers sequences

 primers

	Gene name and symbol	Forward primer	Reverse primer
	Heat shock protein 70kDa (Hsp70)	GGCCACATTGTTGATACATGC	CTACAGTGCAACCACCATGC
	Heat shock protein 27kDa (Hsp27)	AATGGTGATCTCCGCTGACT	CCTCTTCCCTATCCCCTGAG
	Proteasome subunit alpha-type 1 (Psma1)	GATCACCGAACCGTAGTTGG	CAACTGTTGCTGAACCTTGC
	Proteasome subunit alpha-type 5 (Psma5)	CCCCACTAATGGAGCCTAGC	AGAGCCAGATTGGACACAGC
	Proteasome subunit beta-type 2 (Psmb2)	TGACTCTCAGCATCCTTGACC	GTTAGGAGTCCCGCTTAGGG
	Proteasome subunit beta-type 4 (Psmb4)	ACTACAAAGATGCCCGTTCG	TTGAGCCAGCTACAGTCACG
	Proteasome subunit beta-type 5 (Psmb5)	GATCAACCCGTACCTTCTGG	CGAGATGCGTTCCTTATTGC
	26S protease regulatory subunit 6B (Psmc4)	CGAGGTGTCCTCATGTATGG	GCAGGTGCATTCTCCTTAGC
	26S protease regulatory subunit 8 (Psmc5)	TAACAAGGTGGACCCTTTGG	TAGAGCAGGACTCCCTTTGG
	26S proteasome non-ATPase regulatory subunit 3 (Psmd3)	CTTTTGAACCTCCTGCTTCG	AGCTGGATAGCCTTGATTCG
	26S proteasome non-ATPase regulatory subunit 4 (Psmd4)	CCTATTCTGGCTGGTGAAGG	GTAGCAATTCCAGCCTCAGC
	26S proteasome non-ATPase
	regulatory subunit 10	TGCACTCCACTCCATTATGC	AGCATCGTAATGGTCCTTCG
	(Gankyrin) (Psmd10)
	Cellular tumor antigen p53 (Tp53)	TGAACCGCCGACCTATCCTTA	GGCACAAACACGAACCTCAAA
	Ubiquitin B (Ubb)	AGTGACGAGAGGCTTTGTCC	ACGAAGATCTGCATTTTGACC
	Ubiquitin C (Ubc)	AGCCCAGTGTTACCACCAAG	ACCCAAGAACAAGCACAAGG
	Glyceraldehyde-3-phosphate dehydrogenase (Gapdh)	TGAAGCAGGCATCTGAGGG	CGAAGGTGGAAGAGTGGGAG
	Hypoxanthine-guanine
	phosphoribosyltransferase	CCTAAGATGAGCGCAAGTTGAA CCACAGGACTAGAACACCTGCTAA
	(Hprt1)
	TATA box binding protein (TBP)	GGCCTCTCAGAAGCATCACTA	GCCAAGCCCTGAGCATAA

Acknowledgements

This work was supported by Cancéropole Grand Ouest and CNRS. SL & FLM were supported by fellowship from the French ministry of higher education and research (MENRT), and FD by fellowship from the "Association pour la Recherche sur le Cancer" (ARC). We thank Dr V.

Mezger (UMR CNRS 7216, Paris, France) for the gift of immortalized MEFs, and V. Noel for

the help in in silico analysis. We thanks too Frédéric Percevault for the establishment of stable clone expressing HSF2 from Hsf2 -/-iMEFs.

Supplementary information is avalaible at the Oncogene website

Analysis of Hsf2 expression in stable clone

For the establishment of HSF2 stably transfected cells, Hsf2 -/-iMEFs were co-transfected with a pIRES Hygro coding for Hygromycin B resistance and either with expression vector PCR3.1 or by PCR3.1 coding for HSF2α. Clones were selected in the medium described in materials and methods supplemented by 0.2 mg / ml of hygromycin B (Calbiochem, Germany). Hsf2 expression was measured at RNA level by RT-PCR and at protein level by HSF2 immunoblot. One million of each selected clone were plated into 10 cm plate and harvested after 24 h of culture. For RT-PCR, total RNA were extracted using Trizol reagent (Invitrogen Life Science, CA USA) according to manufacturer's procedure. Then 5 µg of RNA were retrotrancribed using M-MLV RT (Invitrogen Life Science, CA USA). PCR was performed using Taq DNA polymerase (Qiagen, France) with 100 ng of cDNA and 500 nM of primers. Primers sequences used for Hsf2 are: 5'-CATGTCTAGTGCTGTCCAGC-3' (forward primer) and 5'-GAGCTCATCGACTTCTATGG-3' (reverse primer). Gapdh served as loading control. For HSF2 immunoblot, cells were treated with 1 µM of MG132 for 10 h and nuclear extracts were prepared with the nuclear extract kit (Active Motif, Belgium) in accordance with manufacturer's procedure. 30 µg of protein were loaded and separated on polyacrylamide gel. Proteins were transferred on nitrocellulose membrane and HSF2 were revealed by an anti-HSF2 antibody (sc-13517) (Santa Cruz Biotechnology, CA USA).

Ponceau staining were shown as loading control. Hsf2α expressed this transcript whereas cells transfected with the expression control vector PCR3.1 did not. Slight expression differences between each clone could be the results of the number of insertion of Hsf2 or chromatin compaction in the insertion site. Expression of Hsf2 in WT cells was realized as positive control and showed the two isoforms of Hsf2 α and β.

HSF2 is a constitutive expressed protein and a constitutive degraded proteins. To detect this transcription factor cells must be treated with MG132 to stabilize HSF2. HSF2 immunoblot (B) in cells transfected with PCR3.1 did not show the presence of HSF2 whereas in cells transfected with HSF2 a strong expression was detect in clone 4 and 5. In clone 6 HSF2 expression was found less important compared to other clones. HSF2 immunoblot in WT cells served as positive control.