Figure 6: BCL-2 inhibition causes cells to undergo apoptosis in established adenomas.

(a) Isolation of Apc^+/+ and Apc^−/− cells at day 14 post low-level clone induction in AhCre^ER.Apc^fl/fl.Lox-STOP-Lox-tdTomato^+/− mice. The left panel represents the FACS sort analysis and the graph shows the relative Bcl-2 expression of the Apc^+/+ compared with the Apc^−/− cell population. Error bars represent s.d. (n=3 per condition), ***P<0.001, Student’s t-test. (b) Lgr5-GFP and BCL-2 stained adenomas in a Lgr5.Apc^fl/fl mouse 5 weeks post induction demonstrating overexpression of Bcl-2 in the Lgr5-GFP⁺ adenomatous glandular structures. Scale bars, 100 μm. (c) Representative images of Apc-deficient spheroids; upper image showing a viable spheroid of the control group and the lower image demonstrating a non-viable spheroid as observed after ABT-199 treatment. The graph displays the amount of viable spheroids counted at 72 h after the start of ABT-199 treatment for the indicated doses. Scale bars, 50 μM. Dots indicate replicates (minimal n=5 per condition), error bars represent the s.e.m. *P<0.05, ***P<0.001, ****P<0.0001, Student’s t-test. (d) Lgr5.Apc^fl/fl mice with established adenomas 4 weeks following induction were treated with ABT-199 for 4 consecutive days. Cell death within these adenomas was compared with cell death within adenomas of untreated Lgr5.Apc^fl/fl mice by counting the amount of cleaved caspase-3 (CC-3) positive foci for each glandular structure. Each dot represents a gland (minimal n=62 glands per condition), error bars indicate the s.e.m. ****P<0.0001, Student’s t-test. (e) FACS graphs indicating the sorting gates for AhCre^ER.Apc^+/+ and AhCre^ER.Apc^−/− cells that have lost a JC-1 signal. (f) Bar graph showing the percentage of cells undergoing mitochondrial depolarization for the indicated concentrations of the BID peptide. The percentage of cells undergoing mitochondrial depolarization was measured with JC-1 staining and was calculated relative to the negative (DMSO) and positive (CCCP) control.