Fig. 3: BIK induces discrete foci of DNA damage.

a Western blot analysis after sorting MDA-MB-231 cells displaying mild caspase activation (left) or total cell population. Cells were treated with 250 ng/ml of Dox or STS (2.5 μM) as indicated for 24 h followed by staining with 5 μM of Cell Event Green reporter followed by fluorescence-activated cell sorting. To obtain cells with mild caspase activation, 100,000 cells were collected from the left side cell populations on the FACS histogram by positioning the sorting gate in the center of the peak. For total cell population analysis (right), cells grown and treated in cell-culture plates were directly collected by trypsinization. Floaters were collected in both cases. Cell lysates were prepared in RIPA buffer and 20 μg total protein was resolved on a 12% SDS-PAGE followed by western blotting with the indicated antibodies. Histogram cartoons at the bottom indicate which part of the flow-cytometry histogram was used to collect cells. b Western blot showing siRNA mediated knockdown of BIK expression reduces γH2AX formation. MCF-7 Tet-on cells were pretransfected with either scrambled or two BIK specific siRNAs, and 24 h later stimulated with 50 ng/ml Dox. c Western blot analysis of BIK expression and γH2AX formation in MCF-7 Tet-on cells using indicated amounts of Dox. d Top: Western blot analysis of MCF-7 Tet-on cells expressing either Empty vector, Wt BIK or BIKΔBH3 at the indicated Dox induction for 24 h. Bottom: Densitometric quantitation of three independent western blots was performed using ImageJ. Error bars represent SD. One-way ANOVA followed by Tukey′s post-hoc test was performed to compute significance among groups. e Top: Representative images of comets formed by Empty vector or BIK-expressing MCF-7 cells at the indicated Dox stimulation for 24 h. Bottom: % DNA in the comet tail per nuclei was quantitated using CaspLab comet analysis program. A total of 150 nuclei from three independent experiments were analyzed. An unpaired two-tailed t-test was performed to determine the p-value. Scale bar 100 µm. f Left: Representative images of γH2AX immunofluorescence analysis of BIK-expressing cells. Right: Quantitation of cells with discrete γH2AX puncta representing DNA damage or diffuse staining representing apoptotic morphology. n = 19–20 frames of images with 2569 nuclei for BIK (−Dox), 1652 for BIK (+Dox) and 1693 for Empty vector (+Dox) were analyzed from two biological replicates. One-way ANOVA followed by Sidak′s post-hoc test was performed to compute significance among groups. Scale bar 5 µm. g Quantitation of cells with discrete γH2AX puncta representing DNA damage or diffuse staining representing apoptotic morphology. n = 40 frames of images with 2946 nuclei for BIK (+Dox), 3484 for BIK (+Dox +z-VAD-fmk), and 3804 for BIK∆BH3 (+Dox) were analyzed from four biological replicates. One-way ANOVA followed by Sidak′s post-hoc test was performed to compute significance among groups.