FIGURE 1.

PiT1-depleted cells are more sensitive to TNF-induced apoptosis. A–C, HeLa cells were transiently transfected with an siRNA control (Ct) or an siRNA directed against PiT1. 48 h after transfection, they were either left untreated or treated with 10 μg/ml cycloheximide (C) alone, 50 ng/ml TNF alone (TNF), or 50 ng/ml TNF plus 10 μg/ml cycloheximide (TNF+C) for different times. A, cells were treated for 4 h and then analyzed by Western blot against PARP and tubulin as a loading control. The bands corresponding to full-length PARP (p116) and cleaved PARP (p89) were quantified, and the percentage of cleaved PARP is given below the panels of blots. B, cells were treated with TNF+C for 0, 2, or 4 h and analyzed by Western blot against PARP and tubulin. The bands corresponding to full-length PARP (p116) and cleaved PARP (p89) were quantified, and the percentage of cleaved PARP is given under the panels of blots. C, cells were stained with propidium iodide for the analysis of their DNA content, and the percentage of cells with a sub-G₁ staining was calculated. D, HeLa cell clones stably expressing either a control (Ct) or a PiT1 shRNA (clones 1 and 2) were treated with 50 ng/ml TNF and 10 μg/ml cycloheximide (TNF+C) for 0, 3, or 4 h. Apoptosis induction was detected by Western blot showing the cleavage of PARP. The detection of tubulin was used as a loading control. The percentage of cleaved PARP was quantified and is given below. E, Western blot showing the total amount of PiT1 protein expressed by the different clones. The PiT1 antibody (9) is specific for the human protein and detects mainly two forms of the protein in HeLa cells indicated by an asterisk and a double asterisk, which may differ by the glycosylation status. The numbers given below are the ratio of the intensity of PiT1 bands over tubulin, used as a loading control. F, cells (shCt clone or shPiT1 clone 2) were treated with TNF+C for 4 h and analyzed for PARP cleavage by Western blot as in D. Values are the mean ± S.E. (error bars) of six independent experiments. ***, p < 0.0005. G, shRNA control or PiT1 cells were either left untreated (Ct) or treated with 10 μg/ml cycloheximide alone (C) or 50 ng/ml TNF plus 10 μg/ml cycloheximide (TNF+C) for 4 h. Apoptosis was detected by the cleavage of PARP, and tubulin was used as a loading control. The intensity of the bands was quantified, and the percentage of cleaved PARP is given below the Western blot panels. H, T SV40 immortalized MEFs from a knock-out PiT1 embryo (−/−) or a WT littermate (+/+) were incubated with 50 ng/ml human TNF and 100 μg/ml cycloheximide (TNF+C) or 100 μg/ml cycloheximide alone (C) for 7.5 h. Cells were lysed and analyzed by Western blot for the appearance of the cleaved form of caspase-3 and tubulin as a loading control as well as the amount of IκBα. The numbers given below are the ratio of the intensity of IκBα signal over actin, used as a loading control. I, quantification of the apoptosis induction for four independent experiments performed as in H. Values are the mean ± S.E. ***, p < 0.0005.