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. Author manuscript; available in PMC: 2017 Jan 1.
Published in final edited form as: Nat Med. 2015 Dec 14;22(1):78–83. doi: 10.1038/nm.4010

Figure 2.

Figure 2

ABT263 kills SCs by apoptosis. (a) Representative flow cytometric plots to measure apoptosis (left) and quantitation of the percentage of viable (gate II: PI−annexin V−) and apoptotic (gates III and IV: PI−annexin V+ and PI+annexin V+) (right) cells of WI-38 IR-SC 24 h after treatment with vehicle (Veh), 1.25 µM ABT263, 20 µM Q-VD-Oph (QVD), or the combination of ABT263 and QVD. (b) Quantification of the percentage of viable IR-SC 72 h after treatment with vehicle or ABT263 ± QVD as in a. (c) Quantification of SA–β-gal+ cells (white bars) 10 d after exposure to increasing doses of IR (left) or after increasing periods of time after treatment with 10 Gy IR (right), and the viability of the irradiated cells under these conditions after an additional incubation with 1.25 µM ABT263 for 72 h (gray bars). (d) Representative western blot analysis of procaspase-8 (Procasp-8), cleaved caspase-8 (cCasp-8), RIP1, cleaved RIP1 (cRIP1) and β-actin in NC and IR-SC 24 h after incubation with vehicle or 1.25 µM ABT263 (ABT). (e) Top, representative western blot analysis of procaspase-3 (Procasp-3), cleaved caspase-3 (cCasp-3) and β-actin in NC and IR-SC from d. Bottom, normalized expression of Procasp-3 and cCasp-3 in NC and IR-SC. Because the samples used for the western blots in d,e were the same, the same β-actin blot was used as a control for both panels. A mixture of cell lysates from etoposide- or cytochrome c–treated Jurkat cells was used as a positive control (Ctl) for detection of cCasp-3 and cCasp-8. (f) Normalized expression of BCL-2, BCL-xL, BAK and BAX in WI-38 cells by western blot analysis at increasing times after treatment with IR (10 Gy). (g) Quantification of NC (left) and IR-SC (right) viability 72 h after incubation with vehicle, 2.5 µM ABT199, 0.625 µM WEHI539, or the combination of ABT199 and WEHI539. (h) Quantification of NC and IR-SC viability 72 h after transfection with a control shRNA (Ctl) or shRNAs specific for BCL2, BCL2L1, or both BCL2 and BCL2L1. Throughout, data are means ± s.e.m. of three experiments, except for h (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001; one-way ANOVA for c,f; two-way ANOVA for b,e,g,h.