FIGURE 5.

JNK activity is instrumental in the apoptosis of PiT1-depleted cells. A and B, HeLa cells were transiently transfected with a control siRNA (Ct) or an siRNA against PiT1 (PiT1) or PiT2 (PiT2). 48 h post-transfection, cells were either mock-treated (0) or treated with 50 ng/ml TNF and 10 μg/ml cycloheximide (TNF+C) for 5 h. Cells were preincubated (+SP) (or not) for 30 min with the JNK inhibitor SP600125 (30 μm), and the inhibitor was kept in the medium for the whole time course of the experiment. A, cells were lysed and analyzed by Western blot with antibodies directed against PARP and the phosphorylated forms of JNK (pJNK) and tubulin (as loading controls). B, values are the mean ± S.E. (error bars) of three independent experiments performed as described in A. *, p < 0.05. C and D, PiT1 knock-out (−/−), or WT MEFs (+/+) were preincubated (TNF+C+JNKi) or not (TNF+C) with the specific JNK inhibitor JNKi (5 μm) for 30 min. 50 ng/ml TNF and 100 μg/ml cycloheximide were then added to the medium for 6 h. C, total cell lysates were analyzed by Western blot with antibodies against cleaved caspase-3, phosphorylated JNK, and tubulin as a loading control. D, values are the mean ± S.E. of three independent experiments performed as described in C. *, p < 0.05. E and F, PiT1^−/− and PiT1^+/+ MEFs were preincubated (TNF+C+BI), or not (TNF+C), for 30 min with the JNK inhibitor BI-78D3 (5 μm) and then treated (or not; BI) with 50 ng/ml TNF and 100 μg/ml cycloheximide for 9 h. E, cell lysates were analyzed with antibodies against phosphorylated JNK and cleaved caspase-3. Actin was used as a loading control. F, values are the mean ± S.E. of four independent experiments performed as described in E. **, p < 0.005.