Fig. 2

Loss of NHEJ factors or BRCA1-A complex members confers resistance to topotecan in ATM-deficient cells. a Representative immunoblot images depicting abundance of ABRAXAS, BRCC36, BRCC45, and MERIT40 proteins in Atm^−/−Abraxas^−/−, Atm^−/−Brcc36^−/−, Atm^−/−Brcc45^−/−, and Atm^−/−Merit40^−/− cells as compared to WT, Atm^−/−, and Atm^−/−Xrcc4^−/−cells. Tubulin is used as a loading control. Two independent clones (numbers identifiers below) were used per genotype. b Quantification of clonogenic survival assays towards topotecan in Atm^−/−Abraxas^−/− (n = 15), Atm^−/−Brcc36^−/− (n = 15), Atm^−/−Brcc45^−/− (n = 15), Atm^−/−Merit40^−/− (n = 15), and Atm^−/−Xrcc4^−/− (n = 15) cells as compared to WT (n = 15) and Atm^−/− (n = 15) cells. c Representative immunoblot images depicting abundance of ATM, LIG4 (- indicates LIG4, while * indicates an antibody cross-reacting protein) and XRCC4 proteins in Atm^−/−Lig4^−/− and Atm^−/−Xrcc4^−/− cells as compared to WT and Atm^−/− cells. Ku80 is used as a loading control. d, e Crystal violet cell viability assay (d) and quantification of clonogenic survival assays (e) indicating suppression of Atm^−/−- dependent (n = 9) hypersensitivity to topotecan in Atm^−/−Lig4^−/− (n = 9) and Atm^−/−Xrcc4^−/− (n = 9) cells as compared to WT cells (n = 9). Note that Lig4^−/− (n = 9) or Xrcc4^−/− (n = 9) single mutants do not exhibit increased topotecan resistance. f Quantification of clonogenic survival assays showing that Atm^−/−Xrcc4^−/− (n = 6), Atm^−/−Xlf^−/− (n = 10), and Atm^−/−Lig4^LD/LD (n = 6) cells are more sensitive to IR than Atm^−/− (n = 6) cells. For all clonogenic survival, curves (left) and AUCs (right) were generated by using GraphPad Prism 7 (see Methods). Bars represent mean ± s.e.m.; ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; NS = not significant (p > 0.05); two-tailed Student’s t test following F test to confirm equal variance; df = 4 (three independent experiments; n = 5 in b; n = 3 in e; n = 2 in f for each experiment). Additional supporting data, are presented in Supplementary Figure 3. Data for validation of LIG4 or XRCC4 loss suppressing the hypersensitivity of ATM-deficient cells to topotecan in human RPE-1 cells (using both ATM inhibitor and ATM gene knockouts) are presented in Supplementary Figure 4. Source data are provided as a Source Data file