FIGURE 3.

The initiator caspase, caspase-8, is activated early in PiT1-depleted cells. A–D, HeLa cell clones stably expressing either a control (Ct) or a PiT1 shRNA (PiT1) were treated with 50 ng/ml TNF and 10 μg/ml cycloheximide (TNF+C) for 0, 2, or 3.5 h. Cells were lysed and analyzed by Western blot with an antibody recognizing all forms of caspase-8 (full-length (p57) and cleaved (p43 and p18)) (A) and RIP1 (full-length p78 and cleaved p36) (C). Tubulin was used as a loading control. B and D, cells were incubated with TNF+C for 3.5 h and analyzed as described in A and C, respectively. Values are the mean ± S.E. (error bars) of three independent experiments. ***, p < 0.0005; **, p < 0.005. E and F, immortalized PiT1^−/− (−/−) and WT (+/+) MEFs were incubated with 50 ng/ml TNF and 100 μg/ml cycloheximide (TNF+C) for 0, 4, or 7 h. Cells were lysed, and samples were analyzed by Western blot with an antibody against RIP1, cleaved caspase-3, or tubulin as a loading control. F, MEFs were treated with TNF+C for 7 h and analyzed as described in E. Values are the mean ± S.E. of five independent experiments. *, p < 0.05.