Cell death and/or cell survival determination Twenty-four hours after transfection, cells were seeded in 96- wells plates (7.56103/well). Twelve hours later, cells were treated for 18 h with staurosporine (Sigma, St Louis, MO). After treatment, the culture medium was discarded and the remaining viable cells were rinsed twice with PBS buffer and stained for 15 min with 0.5% crystal violet in 50% methanol. Afterward, plates were rinsed and dried. Thereafter, the stained cells were solubilized using 0.1 M sodium citrate (pH 5.4) in 20% methanol. The absorbance of each well was read at 570 nm with a Wallac 1420 Multilabel Counter (PerkinElmer, Courtabœuf, France). The percentage of cell survival was based on the ratio of the relative absorbance of the different samples to that of untreated cells [24,25]. Cell proliferation was determined using the WST-1 assay, which requires a 4 h incubation of cells with the tetrazolium WST-1 salt (4-[3-(4-Iodophenyl)-2-(4-Bonitrophenyl)-2H-5-tetrazolio]-1,3- benzene disulfonate) (Roche, Basel, Switzerland) (10 ml/well) followed by absorbance measurement at 450 nm. The percentage of cellular proliferation was calculated based on a control absorbance. Cell cycle analysis was realized as previously described [26]. Briefly, cells were fixed in 70% ethanol, then treated with RNAse A, stained with propidium iodide and analyzed by flow cytometry. Immunofluorescence analysis Cells growing on glass cover slips were fixed for 5 min with freshly prepared 4% para-formaldehyde, pH 7.0. Cells were then permeabilized for 5 min in PBS containing 0.1% Triton 6100. The cover slips were incubated for one hour with monoclonal anti- Hsp27 antibody (Stressgen, San Diego, Ca). After washing, cover slips were further incubated for one hour with FITC-conjugated goat anti mouse immunoglobulin. Actin detection was performed using Alexa FluorTM 488 Phalloidin (Alexa Fluor, Raleigh, NC). Hoechst staining was used to enumerate nuclei and analyze their morphology. Microphotographs were realized using appropriate filters with Zeiss Axioskop microscope equipped with a 636 lens and a digital camera device. Results shRNA targeting of Hsp27 stimulates staurosporine- mediated apoptosis of two cancer cell lines HeLa cells were transiently transfected with pCI-Neo vector (pCI-Neo), pSuperNeo-ScRNA27 (scramble, Sc27), pSuperNeo- MsRNA27 (mismatch, Ms27) and pSuper-ShRNA27 (Sh27). 48 h after transfection, the level of Hsp27 was determined by western blot analysis. As shown in figure 1.A, Sh27 strongly reduced Hsp27 expression whereas the control RNAs did not. Hsp27 is characterized by its strong anti-apoptotic activity, we therefore analyzed whether a decreased level of Hsp27 could sensitize cells to apoptosis. For this, Sh27 depleted cells were treated 18 h with different concentrations of the kinase inhibitor staurosporine. A cell death was increased in Sh27 cells after a 0.075 mmol/L treatment in comparison to control cells (Fig. 1.B). A similar observation was made in transiently transfected MCF-7 cells (data not shown). We also analyzed the activation of caspase-3, characterized by the generation of p12 and p17 fragments from inactive pro-caspase-3 precursor. Immunoblot analysis was performed after 3 h of treatment at different concentrations of staurosporine. A higher level of processed p17 fragment was noticed in Sh27 cells compared to control Ms27 cells. This confirms our preceding observations, using an anti-sense strategy STAT2, Caspase3 and HDAC6 as Hsp27 Client Proteins [4,8], that depletion of Hsp27 stimulates caspase-3 activity and apoptosis of HeLa cells (Figure 1.C). Hsp27 constitutive depletion increases the percentage of cells in G2M and modulates the expression of pro- and anti-apoptotic polypeptides To analyze the consequences of a constitutive depletion of Hsp27, we next produced HeLa and MCF-7 cells stably transfected with the Sh27 vector. Independent clones were isolated and the level of Hsp27 was analyzed (Figure 2.A). HeLa clones HSh27-1.10 and HSh27-2.2 were depleted up to 70% and 95%, respectively; whereas, the level of Hsp27 in MCF-7 clones MSh27-1.3 and MSh27-2.1 was similarly decreased of only about 60%. The lower efficiency observed in MCF-7 cells may result of the ten fold higher level of Hsp27 in these cells compared to HeLa cells (Fig. 2.A) [27]. As it is well referenced that targeting one member of the Hsps family could modify the expression of other Hsps [20], their levels were investigated. As seen in Fig. 2.A, the constitutive depletion of Hsp27 did not induce significant modifications of Hsp70 and Hsp90 levels in either HeLa or MCF-7 cells. Since Hsp27 has been referred as a survival protein [28], the levels of other proteins in this family as well as some members of the apoptotic machinery were also analyzed. As shown in Fig. 1.B, the depletion of Hsp27 induced important changes in the level of several pro- and anti-apoptotic members of the BH3 family. Survivin, an anti-apoptotic BH3 mimetic protein was over-expressed in Hsp27 depleted HeLa cells; its increased level was inversely correlated to the decrease in Hsp27 level. In contrast, the level of survivin was not altered in MCF-7 clones. The reverse phenomenon was detected for Bcl2. This protein had an increased level in Hsp27 partially depleted MCF-7 cells but not in HeLa cells. In both types of cells, the level of BclXS/L was not modified. In contrast, the pro-apoptotic proteins Bax and Bid were up regulated in both cell lines (Figure 2.B). Hence, cell adaption to Hsp27 depletion correlates with drastic changes in several anti- and pro- apoptotic polypeptides. To further characterize the consequences of Hsp27 depletion, a WST-1 test was performed to determine the efficiency of cell proliferation. Intriguingly, the proliferation index was decreased in HeLa and MCF-7 cells lines under-expressing Hsp27. A 10% (HSh27-1.10) and 20% (HSh27-2.2) decrease in the proliferation index of HeLa clones was detected. The two clones of MCF-7 cells showed a 10% decrease (Fig. 2.C). In order to determine whether the reduced proliferation was linked to cell cycle modifications, exponentially growing cells were propidium iodide stained and analyzed by flow cytometry. In both types of Hsp27 depleted cell lines, the percentage of cells in G2/M phase was increased (Fig. 2.D). 26.8% of HSh27-2.2 cells were in G2/M in contrast to only 16.9% in the control cell line. In depleted MCF-7 clones a similar 5.3% increase in G2/M phase was observed. Furthermore, in Hsp27 depleted cells, we detected an increase in the level of sub-G1 cells. 7.4% were detected in Sh27 cells versus 1.9% in control HeLa cells. A similar phenomenon was observed in MCF-7 cells with an increase from 1.7% to 3.5%. A phenomenon which suggests a slight increase in the ability of cells to commit spontaneous apoptosis. Hsp27 is essential to maintain cytoskeleton integrity through an HDAC6 dependent mechanism To better characterize the reduced proliferation efficiency observed in Hsp27 depleted cells, we analyzed the morphology of the different cells lines. HeLa cells were first stained with Hoechst to analyze nucleus morphology. As shown in Fig. 3.A, Hsp27 PLoS ONE | www.plosone.org 3 January 2012 | Volume 7 | Issue 1 | e29719