Antitumor effect of ACF and ABT-263 combination against TNBC cells. (A) HS578T cells were treated with indicated concentrations of ACF and/or ABT-263 for 24 h. Caspase-3/7 activity was analyzed using Muse Caspase-3/7 kit, as described in the Materials and methods. A total of 4 populations of cells were distinguished: Live [caspase-3/7(-)/7-AAD(-)], apoptotic [caspase-3/7(+)/7-AAD(-)], apoptotic/dead cells [caspase-3/7(+)/7-AAD(+)], and necrotic [caspase-3/7(-)/7-AAD(+)]. (B) Cells were treated with ABT-263 (500 nM) and ACF (2 µM) for 24 h and the expression of MCL-1 was evaluated using western blot analysis. (C and D) MDA-MB-231 and HS578T cells were treated with indicated concentrations of ACF and ABT-263 and cell viability was determined using the sulforhodamine B assay. (E) MDA-MB-231 and HS578T cells were treated with ACF (5 µM) and/or ABT-263 (1 µM) for 6 h and the expression of caspase-8 and caspase-9 and their cleaved forms was detected using western blot analysis. ACF, acriflavine; 7-AAD, 7-amino-actinomycin D.