Figure 4.
Mcl-1 disappears in the early stages of apoptosis induced by other DNA-damaging agents. (A) Mitochondria and S100 were isolated from HeLa cells that were left untreated (lanes 1,5), treated with 100 μM Etoposide (Sigma; lanes 2-4), or treated with 100 Gy of γ-irradiation (lanes 6-9) and harvested for cytosol and mitochondria at the indicated times. The levels of cleaved caspase-3, cytochrome c and Mcl-1 were measured in S100. Mcl-1 mitochondrial levels were compared between the samples. The asterisk denotes a cross-reactive band that indicates equal loading. (B) Elimination of Mcl-1 by RNAi accelerates UV-induced apoptosis. HeLa cells were pretreated with Luciferase or Mcl-1 siRNA as described in Materials and Methods. Triplicate samples were harvested without treatment (UT) or at different time points after UV treatment. Whole-cell lysate prepared with 0.5% CHAPS was used to determine the levels of Mcl-1 and Bcl-xL by Western blot and caspase-3 activity by a fluorogenic assay as described in Materials and Methods.