FIGURE 4.

JNK sustained activity is increased in PiT1-depleted cells. A and B, 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 the phosphorylated forms (pJNK) of all JNK isoforms and genes (p46 and p54). The apoptosis progression was followed by the appearance of the cleaved form of PARP. B, cells were treated or not with TNF+C for 3.5 h and analyzed as described in A. Values are the mean ± S.E. (error bars) of six independent experiments. ***, p < 0.0005. C and D, HeLa cells were either transiently transfected with an siRNA control or directed against PiT1 or PiT2. 48 h post-transfection, the cells were either left untreated (0) or treated with 50 ng/ml TNF and 10 μg/ml cycloheximide (TNF+C) for 5 h. Apoptosis progression was followed by the cleavage of PARP, and phosphorylated JNK was detected by Western blot (pJNK). D, values are the mean ± S.E. of four independent experiments performed as described in C. **, p < 0.005. E and F, PiT1^−/− and PiT1^+/+ MEFs were incubated with 50 ng/ml TNF and 100 μg/ml cycloheximide (TNF+C) for 0, 3.5, or 7 h. Cell lysates were analyzed with antibodies against phosphorylated JNK and cleaved caspase-3. Tubulin or actin was used as a loading control. F, cells were treated with TNF+C for 7 h and analyzed as described in E. Values are the mean ± S.E. of four independent experiments. *, p < 0.05; **, p < 0.005.