Figure 4: ARID1A-deficient cells depend on a G2/M checkpoint due to a chromosomal decatenation defect.
From: ATR inhibitors as a synthetic lethal therapy for tumours deficient in ARID1A
(a) Histogram of the cellular DAPI-stained DNA content, determined by FACS, in asynchronous HCT116 ARID1A+/+ (blue) and ARID1A−/− cells (red). Bar chart illustrating the percentage of cells in G2/M phase of asynchronously growing HCT116 ARID1A+/+ and ARID1A−/− cells. Asterisk indicates statistical significance (P=0.033, Student’s t-test), error bars represent s.d., from four independent experiments. (b) Histogram of the cellular DAPI-stained DNA content, determined by FACS, in HCT116 ARID1A+/+ and ARID1A−/− cells at the indicated time points following release from double thymidine synchronization in G1/early S phase. (c) Bar chart illustrating the percentage of mitotic cells with phosphorylation of Histone H3 on Serine 10. Where shown, HCT116 ARID1A+/+ cells were transfected with siARID1A. 48 h later cells were fixed and stained with FITC-P-S10 Histone H3 and PI. Mitotic cells (P-S10 positive and 2 N) were quantified by FACS. Asterisk indicates a statistically significant difference by Student’s t-test between the indicated comparisons. (d) Western blot of nuclear and chromatin-bound TOP2A. The indicated cell lines were transfected with siRNA targeting ARID1A or control siRNA. Forty-eight hous later, subcellular fractions were isolated and resultant western blots immunoblotted for the indicated proteins. Total amount of TOP2A was quantified and normalized to ARID1A+/+ control siRNA, in each fraction. (e) Bar chart illustrating elevated level of anaphase bridges in HCT116 ARID1A−/− cells compared with ARID1A+/+. A minimum of 50 anaphases were scored in three biological replicate experiments. * P=0.038, Student’s t-test. (f) Western blot of nuclear and chromatin-bound TOP2A from OCCC cell lines, with the indicated ARID1A status. Fractionation was performed as per (d). (g) Three hundred eighty four-well plate cell survival data from HCT116 cells transfected with siRNA targeting TOP2A (red) or siCon (blue). Twenty four hours after transfection, cells were exposed to VE-821 for 5 continuous days. Error bars represent s.d. Survival curves siTOP2A versus siCon P value <0.001, ANOVA. (h). Three hundred eighty four-well plate cell survival data from HCT116 ARID1A+/+ and HCT116 ARID1A−/− cells transfected with siRNA targeting TOP2A (red) or siCon (blue). Viability was estimated 5 days after transfection. Error bars represent s.d. and asterisks indicate a statistically significant difference (P<0.001) by Student’s t-test between the indicated comparisons. FACS, fluorescence-activated cell sorting.
