Figure 4.

A, T98G glioblastoma cells were treated for 72h under reduced serum conditions (1.5% FBS) with CP-d/n-ATF5-S1 (100 μM), ABT263 (0.5 μM) or GX15-070 (50 nM) at the indicated combinations prior to performing MTT assays. Columns: means. Error bars: standard deviation (SD). B, Representative microphotographs at 20x magnification of T98G glioblastoma cells treated with CP-d/n-ATF5-S1, ABT263, the combination of both or solvent for 48h. In addition, microphotographs of cells treated with mutated CP-d/n-ATF5-S1 alone or combined with ABT263 (0.5 μM) are shown. C, Representative flow plots of T98G, LN229, SF188 (pediatric), NCH644 (glioma stem-like) glioblastoma and PANC-1 pancreatic carcinoma, A375 melanoma, K562 chronic myeloid leukemia (in blast crisis) cells that were treated for 72h with CP-d/n-ATF5-S1 (100 μM), ABT263 (0.5 μM), the combination of both or solvent prior to staining with annexin V/propidium iodide and flowcytometric analysis. D, Representative flow plots of T98G glioblastoma cells subjected to treatment with mutated CP-d/n-ATF5-S1 alone or in combination with ABT263. E, Quantitative representation of the fraction of annexin V and/or PI-positive cells treated as described for C. * signifies a p-value of less than 0.05. Columns, means of three serial measurements. Bars, SD. F, T98G glioblastoma cells were treated with CP-d/n-ATF5-S1 (100 μM), mutated CP-d/n-ATF5-S1 (100 μM) and ABT 263 (0.5 μM) at indicated combinations for 48h under serum starvation. Whole-cell extracts were examined by Western blot for caspase-9 (CP9, CF=cleaved fragment), Mcl-1, Bcl-2 and Bcl-xL. Actin Western blot analysis was performed to confirm equal protein loading. Densitometric analysis was performed using ImageJ (National Institutes of Health, U.S.A., http://imagej.nih.gov/ij). Data were normalized first to the respective actin control and second to the respective treatment control.