Figure 8
Bcl-2 K/D is less effective than Bcl-2 in protecting against STS-induced apoptosis in stable WEHI7.2 cell lines. (a) Stable WEHI7.2 cell lines expressing Bcl-2 or Bcl-2 K/D were created. The expression levels of Bcl-2 and Bcl-2 K/D were examined by western-blotting analysis using an anti-Bcl-2 antibody. Control WEHI7.2 cells display very low endogenous Bcl-2 levels. (b) The cleavage of poly(ADP-ribose)-polymerase (PARP) in control WEHI7.2 cells, WEHI7.2 cells expressing Bcl-2 and WEHI7.2 cells expressing Bcl-2 K/D was monitored by western-blotting analysis using an anti-PARP antibody. (c) FACS analysis of untreated and STS-treated PI/Annexin V-FITC-stained control, Bcl-2-overexpressing and Bcl-2 K/D-overexpressing WEHI7.2 cell lines (10 000 cells per analysis). The apoptotic population was identified as the AnnexinV-FITC-positive fraction (P2+P3). The STS-induced apoptotic population was determined by the difference between the Annexin V-FITC-positive fraction of STS-treated cells and the untreated cells: (P2+P3)STS–(P2+P3)untreated. (d) Quantitative analysis from five independent experiments of the STS-induced apoptotic cell population, normalized to the values obtained for the control WEHI7.2 cells (% of control). (e) Fluorimetric analysis from three independent experiments of the caspase-3 activity in untreated and STS-treated control, Bcl-2-overexpressing and Bcl-2 K/D overexpressing WEHI7.2 cells using a plate-reader assay. The difference in relative fluorescence units between STS-treated and untreated cells was calculated and plotted. In all these experiments, both Bcl-2 and Bcl-2 K/D significantly (*) protected against STS-induced apoptosis, but Bcl-2 K/D was significantly ($) less potent than Bcl-2. Statistically significant differences are indicated as in Figure 5
