Fig. 4: Methylation of USP11 at R433 is required for homologous recombination-mediated repair of DSBs. | Nature Communications

Fig. 4: Methylation of USP11 at R433 is required for homologous recombination-mediated repair of DSBs.

From: Arginine methylation and ubiquitylation crosstalk controls DNA end-resection and homologous recombination repair

Fig. 4

a MCF7 cells stably expressing USP11 or USP11-R433K were transfected with siUSP11, exposed to increasing doses of olaparib and cell viability determined by colony survival assay (mean ± SD; n = 3 independent biological repeats; Student’s t test (two-sided, equal variance) *p = 0.010, **p = 0.00257 comparing USP11 with USP11-R433K). b HeLa-USP11 and HeLa-USP11-R433K cells were transfected with siUSP11, exposed to increasing doses of olaparib and cell viability determined by colony survival assay (mean ± SD; n = 3 independent biological repeats; Student’s t test (two-sided, equal variance) **p = 0.006 (1 μM); *p = 0.0412 (2 μM); *p = 0.0539 (3 μM)). c–e MCF7 cells stably expressing doxycycline-inducible shUSP11 and ectopic USP11 or USP11-R433K were treated with doxycycline for 72 hrs, exposed to 3 Gy IR, and harvested at the time points indicated (mean ± SD; n = 3 independent biological repeats). Mitosin or CENPF-positive cells were scored for: c 53BP1 (Student’s t-test (two-sided, equal variance): *p = 0.04, ***p = 0.0005); d RAD51 (Student’s t test (two-sided, equal variance): *p = 0.02 NT versus shUSP11; *p = 0.04 shUSP11/USP11 versus shUSP11/USP11-R433K); e RPA (Student’s t test (two-sided, equal variance): *p = 0.03 NT versus shUSP11 (2 h post IR); *p = 0.02 shUSP11/USP11 versus shUSP11/USP11-R433K (2 h post IR); p = 0.05 NT versus shUSP11 (6 h post IR); *p = 0.03 shUSP11/USP11 versus shUSP11/USP11-R433K (6 h post IR)). Scale bar represents 5 μm. f HeLa cells stably expressing USP11, USP11-R433K or USP11-C318S were transfected with siUSP11, exposed to 3 Gy IR, and harvested 6 hrs later. CENPF-positive cells were scored for RPA foci formation (mean ± SD; n = 3 independent biological repeats, **p = 0.0057 (Student’s t test (two-sided, equal variance); ****p < 0.0001; (ordinary one-way ANOVA and Tukey post hoc test)). g PRMT1 and USP11 are epistatic in the regulation of RPA foci formation after IR (3 Gy) in HeLa late S/G2 cells (mean ± SD; n = 3 independent biological repeats; ****p < 0.0001; one-way ANOVA and Tukey post hoc test). Uncropped blots, raw and processed graphical data provided as a Source Data file.

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