STAT2, Caspase3 and HDAC6 as Hsp27 Client Proteins Figure 5. Endogenous level of STAT2 is decreased in Hsp27 depleted cell line. A, Hsp27 depleted and control HeLa cells were collected, and samples were analyzed by immunoblotting using antibodies that recognize the different STAT polypeptides. Quantification of STA2 level was reported in a table. B, as in A) but analysis of STAT2 level in MCF-7 depleted cells. C, Analysis of STAT3 phosphorylation. HeLa clones were treated with interleukin-6 (4 h; 125 ng/mL) or heat shock (60 min; 42uC and 44uC). Samples were collected and analyzed by immunobloting with STAT3, Phospho- STAT3, Hsp27 and actin antibodies. D, quantification of stat2 gene relative expression by qPCR. E, Effect of proteolytic inhibitors. HeLa cells were analyzed after being treated with MG132 or ALLN, as described in Fig. 3. Level of STAT2 was revealed by immunobloting. F, HeLa cells were transiently transfected with an ISRE reporter gene construct [36] and treated for 6 h with 50 U/mL of interferon-a. They were also treated or not for 30 min with 10 mM of either SB203580 or SB202190 (p38MAPkinase and MK2 inhibitors, respectively) before exposure to interferon-a. Luciferase expression correlated with STAT2 transcriptional activity was quantified in both cell lines. doi:10.1371/journal.pone.0029719.g005 fractions that were positive for the targetted proteins (native sizes of about 100–200 kDa in the case of procaspase-3, 250–500 kDa in the case of STAT2 and 550–650 kDa in the case of HDAC6). Immunoblots were revealed with the corresponding antibody (Fig. 6.B). These results demonstrate a strong interaction between the three tested polypeptides and Hsp27. Interactions were restrained to sub-fractions of total Hsp27 that are characterized by a specific level of oligomerization. Hence, these interactions should highly correlate with specific structural organizations of the different partners. However, we cannot exclude that the medium and large populations could also include interacting complex formed by HspB1 small oligomers with several protein partners. Discussion In the present study, we characterized cancerous HeLa and MCF-7 human cells lines that show reduced level of Hsp27 expression consequently of the transient or constitutive expression of shRNA targeting Hsp27 mRNA. Isolation of Hsp27 depleted stable clones was a rather difficult task since few cells survived the selection, a phenomenon that could be linked to the lack of Hsp27 mediated protective activity. We noticed in the survival clones that the constitutive knock down or reduced levels of Hsp27 did not modulate the levels of major Hsps, as Hsp70 and 90. In contrast, pro-apoptotic proteins like Bax and Bid were over-expressed in both HeLa and MCF-7 cells, a possible response towards the increased tendency of cancer cells under-expressing Hsp27 to undergo spontaneous apoptosis [4,8,17,21]. On the other hand, over-expression of survival proteins like survivin (HeLa cells) or Bcl-2 (MCF-7 cells) could result of adaptation mechanisms to survive in the presence of low levels or absence of Hsp27. Of interest, the constitutive knock down of Hsp27 induced the degradation of HDAC6, STAT2 and procaspase-3 proteins without down regulating their mRNA transcripts levels. Further- more, we demonstrated a biochemical interaction between different oligomeric forms of Hsp27 and these specific polypep- tides. This highly suggests a new molecular chaperoning function of Hsp27 towards native polypeptides. Additionally, the slow growing HeLa clones, able to adapt to Hsp27 knock-down, displayed nucleus abnormalities and polyploidy. PLoS ONE | www.plosone.org 8 January 2012 | Volume 7 | Issue 1 | e29719