Fig. 3
Combination-induced cell death occurs without caspase-3 activation and disruption of the mitochondrial membrane potential. A Western blot analysis of cleaved caspase-3 in NCH82 cells treated with the vehicle control (DMSO), 15 µM Camptothecin (CPT) and the combination of 48 µM TMZ and 2.66 µM SKI-II (TMZ + SKI-II). Full-length blots are presented in Additional file 6. (B, Bi) Flow cytometric quantification of active caspase-3 FITC-positive NCH82 cells treated with the vehicle control (DMSO), 1 µM Camptothecin (CPT) and (TMZ + SKI-II) for 72 h under normoxia (21% O2) and hypoxia (3% O2). B Representative plots of NCH82 cells treated under hypoxia. (Bi) Analysis and quantification of the data was performed with the software FlowJo. Data are expressed as the mean (± SD) of the percent of total single cells; n = 3. One-way ANOVA followed by Tukey’s multiple comparisons test was performed for statistical analysis. Analysis of the mitochondrial membrane potential by TMRE labelling in NCH82 treated cells via (C) live cell fluorescence microscopy after 48 h at 21% O2 and (D) flow cytometry after 24- to 48 h under normoxia and hypoxia. Treatment with the depolarization control FCCP (50 µM) for 60 min. The median fluorescence intensity (MFI) of TMRE was calculated with FlowJo. Data represent mean (± SD) of n = 3. Two-way ANOVA followed by Tukey’s multiple comparisons test was performed for statistical analysis. Scale bar = 200 µm