STAT2, Caspase3 and HDAC6 as Hsp27 Client Proteins Figure 2. Hsp27 knock down clones, enriched in G2/M cells, show decreased cell proliferation. Constitutive Hsp27 depleted clones of HeLa or MCF-7 were isolated as described in Materials and methods. A, HeLa or MCF-7 samples from independent Hsp27 depleted clones were collected and levels of Hsp27, 70 and 90 were analyzed by western blot. B, Immunoblot analysis of Hsp27, survivin, Bcl-2, BclXs/l, Bax, Bid and actin present in both HeLa and MCF-7 depleted clones. C, WST-1 test was performed each 12 h during five days. Proliferation index was determined and reported for each cell line (p,0.01). D, Cell cycle study was performed by flow cytometry analysis after PI staining as previously described [26]. Quantifications experiments were realized and reported in tables presented on the left of the flow cytometry analysis. doi:10.1371/journal.pone.0029719.g002 proteolysis inhibitors, such as the proteasome inhibitor MG132 or the calpain inhibitor ALLN. In our cells, MG132 was able to block the proteasomal pathway since the phenomenon correlated with poly-ubiquitin accumulation (Fig. 3.F). Both ALLN and MG132 up-regulated the level of HDAC6 in HSh27cells (Fig. 3G). Consequently, similar levels of HDAC6 were observed in every HeLa cells lines. This suggests that an increased degradation of HDAC6 occurs in the absence of Hsp27. Endogenous level of pro-caspase-3 is decreased in Hsp27 depleted cells Hsp27 has been described to interact with the pro-domain of procaspase-3, a key protease involved in the executive pathway of apoptosis [32]. Physical interaction with Hsp27 inhibits procas- pase-3 processing leading to the activation of apoptotic caspases and participates to the mechanism of cell survival mediated by Hsp27 [7]. As shown in the immunoblots presented in Fig. 4 panel A, the level of endogenous procaspase-3 was down regulated in Hsp27 depleted HeLa clones. Quantification analysis revealed a 79.6% decrease in pro-caspase-3 level in the most Hsp27 depleted clone HSh27-2.2. As observed in transitory experiments, process- ing of procaspase-3 in p12 and p17 fragments after a 0.2 mmol/L staurosporine treatment was increased in all Hsp27 depleted HeLa clones (Figure 4B). In contrast, p12 and p17 cleaved fragments were not observed in the absence of apoptotic stimulation (data not shown) allowing us to conclude that the decrease in procaspase-3 level was not due to an increased procaspase-3 processing. To investigate if the reduced level of procaspase-3 protein originated from an inhibition at the gene transcription level, procaspase-3 gene product was quantified by qPCR (Fig. 4.C). Procaspase-3 mRNA was up regulated by 50% in HSh27-2.2 clone, indicating an increased in transcriptional activity when compared to control cells. As a consequence, these data strongly suggest a high rate of degradation of the protein. Thus, we tested procaspase-3 protein level when proteasomal and calpain pathways were inhibited by MG132 and ALLN, respectively (Fig. 4.D). Procaspase-3 level was drastically increased during MG132 treatment but was not restored by ALLN, suggesting an ubiquitin/proteasomal degradation in cells depleted of Hsp27. The analysis was not performed in MCF-7 cells because of the lack of expression of procaspase-3 in these cells. PLoS ONE | www.plosone.org 5 January 2012 | Volume 7 | Issue 1 | e29719