Figure 7.

Role of Bax. (a) A-375 and A-375-TS were treated for 1 h with TRAIL (20 ng/ml)±BMS-345541 (A-375, 2 μM; A-375-TS, 5 μM) and were analyzed for Bax conformational changes by flow cytometry (Bax-NT). (b) Total protein extracts (caspase-8) and mitochondrial extracts (tBid and Bax) of A-375 and A-375-TS treated for 2 h were investigated by western blotting. Equal loading was proven by GAPDH and the mitochondrial protein VDAC, respectively. (c) A-375 and A-375-TS were treated for 1 h with BMS-345541 (2 μM/5 μM) and were analyzed by flow cytometry for total Bax, phosphorylated (p)Bax(Ser167) and pBax(Thr184) expression. Below, signals are shown for HCT-116 and HCT-116 Bax-KO cells. (d) A-375 and A-375-TS, treated with the same conditions, were analyzed after 24 h. (e) Protein expression (western blotting) of endogeneous I-κBα (39 kDa) and I-κBα-SR (45 kDa) is shown in mock- and I-κBα-SR-transfected A-375 cells at 24 h. (f) Relative p65 DNA-binding capacity is shown in nuclear extracts of mock- and I-κBα-SR-transfected A-375 cells, as quantified by enzyme-linked immunosorbent assay (ELISA) (24 h after transfection, 1 h treatment with TNF-α). (g) Apoptosis and cytotoxicity were monitored in mock- or I-κBα-SR-transfected A-375 treated in addition with BMS-345541 and/or TRAIL. Treatment with BMS-345541/TRAIL (for 24 h) started at 24 h after transfection. Mean values and S.D.'s of a representative experiment are shown (one of two, each with triplicates). (h) I-κBα-SR- or mock-transfected A-375 cells were analyzed by flow cytometry at 24 h for Bax conformational changes (Bax-NT) and for Bax phosphorylation by pBax(Ser167) and pBax(Thr184) antibodies. For control, A-375 cells were treated in parallel for 24 h with BMS-345541 (2 μM) and are shown below. For all flow cytometry data, treated cells are shown in overlays with respective non-treated controls and/or IgG1-stained controls. Always two independent experiments with triplicates revealed highly comparable results