Figure 1.

UV induces cytochrome c release from HeLa cells in vivo and in vitro. (A) Mitochondria and S100 were harvested from HeLa cells treated with UV for different amounts of time as described in Materials and Methods. In lanes 5-8, we preincubated the cells in the presence of Z-VAD.fmk, a broad-spectrum caspase inhibitor as indicated. Cytochrome c, caspase-9, and caspase-2 were analyzed in S100. Bak oligomerization was analyzed in mitochondrial fractions as described (Wei et al. 2000). (B) Mitochondria from UV-treated cells release cytochrome c in vitro. Mitochondria (0.67 mg/mL) from untreated cells (lane 1) or cells that were UV-treated for 30 min (lane 2), 60 min (lane 3), or 120 min (lane 4) were incubated in vitro at 37°C for 30 min. Following incubation, the mitochondria were pelleted and analyzed as described in Materials and Methods. (C) S100 inhibits cytochrome c release from primed mitochondria. Mitochondria (0.67 mg/mL) from cells treated with UV for 1 h were coincubated (lanes 1-5) at 37°C for 30 min with only buffer (lane 1) or HeLa S100 (4 mg/mL) from untreated cells (lane 2) or cells that were UV-treated for 30 min (lane 3), 60 min (lane 4), or 120 min (lane 5). Mitochondria and supernatants were analyzed for Bak oligomerization and cytochrome c release, respectively.