Fig. 2: BIK induces sublethal apoptosis with DNA damage.

a Quantitation of relative mean fluorescence intensity after stimulating MDA-MB-231 Tet-on cells at the indicated doses of Dox for 24 h followed by staining with 10 μM of CaspACE (FITC-VAD-FMK) and flow-cytometric analysis. Four independent experiments were performed and at least 10,000 cells (5000 for staurosporine) were acquired for all treatments. One-way ANOVA followed by Sidak′s post-hoc test was performed to compute significance among groups. b Western blot analysis of MCF-7 Tet-on cells expressing BIK at the indicated Dox concentrations in the presence or absence of z-VAD-fmk (10 µM) for 24 h. c, d MCF-7 and MDA-MB-231 Tet-on cells were, respectively, stimulated with indicated doses of Dox for 24 h followed by staining with propidium iodide and flow-cytometric analysis. At least 10,000 cells were acquired for three independent experiments. One-way ANOVA followed by Sidak′s post-hoc test was performed to compute significance among groups. e, f Flow-cytometry profiles of MDA-MB-231 Tet-on cells stained with Cell Event Green caspase reporter. Cells were stimulated at the indicated doses of Dox for 24 h followed by staining with 5 μM of Cell Event Green reporter followed by flow-cytometric analysis. Note the rightward shift of the histogram profiles of BIK-expressing cells. 2.5 μM staurosporine was used as a positive control. g Bar graph depicting % caspase-positive cells obtained by quantitation of e, f. Four independent experiments were performed and at least 10,000 cells were acquired for all treatments. One-way ANOVA followed by Sidak′s post-hoc test was performed to compute significance among groups. h Bar graph depicting the quantitation of relative mean fluorescence intensity for the main peak of the histograms shown in e and f. Four independent data points were collected and for all treatments. One-way ANOVA followed by Sidak′s post-hoc test was performed to compute significance among groups. i (Left) Representative images of clonogenic survival assay after sorting MDA-MB-231 cells displaying frank or mild caspase activation. Cells were treated with 250 ng/ml of Dox or STS as indicated for 24 h followed by staining with 5 μM of Cell Event Green reporter followed by fluorescence-activated cell sorting. Thousands cells each were collected directly in cell-culture plates from the left or right side cell populations on the histogram by positioning sorting gates in the center of the respective peaks. Colonies were allowed to form for 8 days. Histogram cartoons on the extreme left indicate which part of the flow-cytometry histogram was used to collect cells. (Right) Bar graph depicting % clonogenic survival of frank or mild caspase-positive cells. Three independent experiments were performed. One-way ANOVA followed by Sidak′s post-hoc test was performed to compute significance among groups.