FIG. 1.

HSP27 enhances proteasome activation while inhibiting apoptosis. (A) U937 cells either were left untreated or were treated for 4 h with 100 μM etoposide (VP16) in the absence or presence of acetyl-calpastatin (25 μM), MG132 (25 μM), or lactacystin (25 μM) before measurement of the ability of cell lysates to cleave the substrate Suc-LLVY-AMC (black bars; AU, arbitrary units) and the percentage of apoptotic cells after nuclear chromatin staining with Hoechst 33342 (gray bars). (B) Control-transfected (white bars) and HSP27-transfected (black bars) cells either were left untreated or were treated for 4 h (U937 cells) or 24 h (MEF cells) with 100 μM etoposide (VP16) or 20 ng of ΤNF-α/ml in the absence or presence of 25 μM MG132 before measurement of the ability of cell lysates to cleave the substrate Suc-LLVY-AMC. Results are expressed as percentages (100% is the activity in VP16-treated, HSP27-transfected cells). Insets show Western blot analyses of HSP27 expression in control- and HSP27-transfected cells. (C) Kinetic analysis of Suc-LLVY-AMC cleavage activity (black bars) and apoptosis induction (gray bars) in control- and HSP27-transfected U937 cells treated with 100 μM etoposide for the indicated times. (D and E) Cells were treated with etoposide (100 μM, 4 h) in the presence of decreasing concentrations of MG132 or lactacystin, as indicated. Then, chromatin staining with Hoechst 33342 was used to measure the percentage of control-transfected and HSP27-transfected U937 apoptotic cells (D) or the percentage of cell survival induced by HSP27 overexpression (E). The percentage of proteasome inhibition (Suc-LLVY-AMC cleavage) is also indicated (E). Data are the means and standard deviations for three independent experiments.