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. 2017 Aug 10;45(18):10614–10633. doi: 10.1093/nar/gkx715

Figure 6.

Figure 6.

DNA-PKcs and ATM are essential for H2AX-dependent NHEJ. (A) Percentage of I-SceI-induced GFP+ cells from DNA-PKcs+/+H2AX–/– and DNA-PKcs–/–H2AX–/– BGN reporter cell clones. Bars represent the mean ± S.D. of three independent experiments, each in triplicates. One-way Anova: P< 0.0001 between ‘DNA-PKcs+/+H2AX–/–’ and ‘DNA-PKcs–/–H2AX–/–’. To generate DNA-PKcs–/–H2AX–/– clones using CRISPR/Cas9, H2AX–/– BGN reporter mouse ES cells were transfected twice with Cas9 and gRNAs targeting DNA-PKcs and then plated on MEF. In ∼2 weeks, individual clones were picked, and DNA-PKcs–/–H2AX–/– clones with identical deletions in two DNA-PKcs alleles were verified by Sanger sequencing of the edited site. DNA-PKcs+/+H2AX–/– clones were also obtained from the same treatment. (B and C) Percentage of I-SceI-induced GFP+ cells from two DNA-PKcs+/+H2AX–/– BGN reporter clones (clone#1 and #3) and two DNA-PKcs–/–H2AX–/– BGN reporter clones (clone#71 and #85) transiently (B) or stably (C) transfected with EV, wtH2AX and/or S139A expression plasmids. Bars represent the mean ± S.D. of three independent experiments, each in triplicates. Student's paired t-test between ‘EV’ and ‘H2AX’ in DNA-PKcs+/+H2AX–/– clones: P = 0.044 in clone #1 and P = 0.026 in clone #3 in transient transfection experiments; P = 0.019 in clone #1 and P = 0.04 in clone #3 in stable transfection experiments; in DNA-PKcs–/–H2AX–/– clones #71 and #85: NS in either transient transfection or stable transfection experiments. Expression of exogenous HA-tagged H2AX and/or its mutant S139A was detected by Western blot as indicated. (D) Percentage of I-SceI-induced GFP+ cells from ATM+/+H2AX–/– and ATM–/–H2AX–/– BGN reporter cell clones. Bars represent the mean ± S.D. of three independent experiments, each in triplicates. One-way Anova: P = 0.014 between ‘ATM+/+H2AX–/–’ and ‘ATM–/–H2AX–/–’. ATM–/–H2AX–/– clones and ATM+/+H2AX–/– were similarly generated by CRISPR/Cas9 gene editing as in (A). ATM–/–H2AX–/– clones with identical deletions in two ATM alleles were verified by Sanger sequencing of the edited site. (E and F) Percentage of I-SceI-induced GFP+ cells from two ATM+/+H2AX–/– BGN reporter clones (clone #3 and #15) and two ATM–/–H2AX–/– BGN reporter clones (clone#37 and #42) transiently (E) or stably (F) transfected with EV, wtH2AX and/or S139A expression plasmids. Bars represent the mean ± S.D. of three independent experiments, each in triplicates. Student's paired t-test between ‘EV’ and ‘H2AX’ in ATM+/+H2AX–/– clones: P = 0.024 in clone #3 and P = 0.037 in clone #15 in transient transfection experiments; P = 0.011 in clone #1 and P = 0.001 in clone #3 in stable transfection experiments; in ATM–/–H2AX–/– clones #37 and #42: NS in either transient transfection or stable transfection experiments. Expression of exogenous HA-tagged H2AX and/or its mutant S139A was detected by Western blot as indicated.