Figure 5.

Hsp27 regulates Fas-induced apoptosis by modulating FADD binding to PEA-15. (a) Hsp27 suppresses Fas-induced apoptosis. LNCaP_mock and LNCaP_Hsp27 cells were treated with either 2.5 μg/ml CHX alone, 1.0 μg/ml agonistic anti-Fas antibody (CH-11) alone or in combination in 5% charcoal-stripped serum media. Apoptosis was assessed by measuring the percentage cells containing subdiploid DNA (% sub-G1) as determined by propidium iodide staining and flow cytometry 24 h after treatment. (b) Hsp27 silencing promotes Fas-induced apoptosis. At 48 h after transfection of LNCaP cells with 20 nM Scr or 20 nM siHsp27, apoptosis in response to CH-11 was assessed as above. (c) Hsp27 regulates association of PEA-15 with FADD. PEA-15 was immunoprecipitated from cell lysates from LNCaP_mock and LNCaP_Hsp27 cells and from LNCaP cells transfected with 20 nM Scr or 20 nM siHsp27 and cultured for 24 h. Immune complexes were resolved on SDS-PAGE and immunoblotted for FADD and PEA-15. (d) PEA-15 silencing protects from Fas-induced apoptosis. LNCaP_mock and LN-221 cells were treated in the presence or absence of 2.5 μg/ml CHX and 1.0 μg/ml CH-11. Apoptosis was assessed 24 h after treatment. (e) Role of PEA-15 in intrinsic apoptosis. LNCaP_mock and LN-221 cells were treated with Scr or siHsp27 in the presence or absence of paclitaxel. Percentage of cells undergoing apoptosis was measured as above. The results (a, b, d and e) are the means of three independent experiments. Values shown represent mean±S.D. The symbol ‘^*' denotes statistical significance (P<0.05)