Fig. 6: Over-expression of BAD leads to the death of colorectal cancer cells (CRCs).

A, B Caco-2 (A) and HT-29 (B) were made to express mCitrine-conjugated BAD (BAD) or BAD mutants harboring S112A and S136A double mutations (BAD-AA). BAD-expressing cells were treated with either R18 (10 µM) or FTY720 (2 µM) for 24 h. The mitochondrial membrane potential sensor TMRE (100 nM) was added to the medium 1 h prior to live-cell imaging. Representative images were selected from three independent experiments, with at least five images were captured per dish in each experiment. C, D The co-localization of BAD and mitochondria in Caco-2 (C) and HT-29 (D) was assessed by calculating Pearson’s correlation coefficient (R). Each data point corresponds to a single cell. E, F Caco-2 (E) and HT-29 (F) cells were transfected with either pBI-14-3-3ζ-Rluc8-mCitrine, pBI-14-3-3ζ-Rluc8-BAD-mCitrine, or pBI-14-3-3ζ-Rluc8-BAD-AA-mCitrine and allowed for 72 h for plasmid expression. Cell death was evaluated using the Operetta CLS system. BAD-induced cell death was quantified as the ratio of YFP- and propidium iodide (PI)-double-positive cells to YFP-positive cells. Data were collected from five independent experiments, each with triplicate measurements. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.