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. Author manuscript; available in PMC: 2014 Oct 10.
Published in final edited form as: Clin Cancer Res. 2011 Sep 20;17(22):7093–7104. doi: 10.1158/1078-0432.CCR-11-0578

Figure 3. The combination of ABT-737 and 4-HPR induced caspase-dependent apoptosis through mitochondrial membrane depolarization and cytochrome c release.

Figure 3

Figure 3

(A and B) CHLA-119, CHLA-15, SMS-KAN or CHLA-136 NB cells were treated with ABT-737, 4-HPR, or the combination for 24 hours (for CHLA-15, 3 hours). Drug concentrations used were 2.5 μM for CHLA-119 and CHLA 15, and 5 μM for SMS-KAN and CHLA-136. (A) Cells were then analyzed for apoptosis (TUNEL assay) by flow cytometry. Bars show the percentages of TUNEL-positive cells, defined as apoptotic. (B) CHLA-119, CHLA-15, and SMS-KAN cells were incubated with the JC-1 mitochondrial probe and analyzed by flow cytometry. Bars show the percentages of mitochondrial membrane-depolarized cells. (C) Cytostolic and mitochondrial extracts from CHLA-119 cells incubated for 6 or 24 hours with ABT-737, 4-HPR or the combination were prepared and immunoblotted with an anti-cytochrome c (cyt.-c) antibody. β-Actin and COX IV were used as the loading control for cytosolic and mitochondrial fractions, respectively. (D - F) CHLA-119 and CHLA-15 cells were incubated with ABT-737, 4-HPR, or the combination for 1.5, 3 hours (CHLA-15) or 6, 24 hours (CHLA-119). (D) Cell lysates were then examined by western blot analysis for caspase-9 and caspase-3. (E) Cell lysates from CHLA-119 (24h) and CHLA-15 (1.5h) were incubated with caspase-8 substrate using a colorimetric assay. Bars show the mean fold increase of casapse-8 activity. (F) CHLA-119 and CHLA-15 cells were pre-treated with Boc-d-fmk (40 μM) for 1 hour before being exposed to 2.5 μM ABT-737, 4-HPR, or the combination for 3 or 24 hours. After treatment, apoptotic cells were measured by flow cytometric TUNEL assay. Bars show the percentage of TUNEL-positive cells, defined as apoptotic. Data shown are representative of two independent experiments. In A, B, E, and F, data represent mean + standard deviations (SD) of triplicate samples. ** p < 0.005, *** p <0.001.