Extended Data Fig. 9: HELQ is epistatic with RAD52 for SSA and additive for second-end capture.
From: HELQ is a dual-function DSB repair enzyme modulated by RPA and RAD51
a, HELQ protein levels in U2OS WT cells, post 72 h post siRNA transfection, confirmed by HELQ-immunoprecipitation and western blot. b, HELQ protein levels in U2OS WT and HELQ–/– cells, confirmed by HELQ-immunoprecipitation and western blot. c, HELQ gene expression after treatment with siRNA post 72 h in U2OS cells. siNT n = 9, siHELQ_1 n = 9, siHELQ_2 n = 3, siHELQ_3 n = 3 independent experiments ± S.E.M, compared to siNT. d, Schematic representation of DR-GFP reporter assay for measuring DSB repair by HR. e, I-SceI-induced HR frequency in U2OS DR HELQ–/– cells and in U2OS-DR cells treated with the indicated siRNA. siNT/WT, n = 20; siHELQ_1, n = 5; siHELQ_2, n = 13; siHELQ_3, n = 10; HELQ–/–, n = 7 independent experiments ± S.E.M, compared to siNT or WT cells. f, BRCA2 and RAD52 proteins levels 72 h post siRNA transfection confirmed by western blot. g, I-SceI-induced SSA frequency in U2OS-SA cells treated with the indicated siRNA. siNT, n = 21; siRAD52, n = 4; siHELQ_1, n =4; siHELQ_2, n = 10; siHELQ_3, n = 3; siHELQ_1/RAD52, n = 4, siHELQ_2/RAD52, n = 8; siHELQ_3/RAD52, n = 3 independent experiments ± S.E.M, compared to siNT. h, Detection of formation of γH2AX, RPA2, and RAD51 foci after 10 Gy IR treatment in U2OS WT and HELQ–/– cells. i, Quantification of RPA2, RAD51 and γH2AX foci in experiments as shown in h. n = 3 independent experiments ± S.E.M, compared to siNT. j, Detection of formation of γH2AX, RPA2, pRPA2 S4/8 and RAD51 foci after treatment with 4 µM CPT (Camptothecin) of U2OS WT and HELQ–/– cells. k, Quantification of γH2AX, pRPA S4/8 foci and RAD51 foci in experiments as shown in j. n = 3 independent experiments ± S.E.M, compared to siNT. l, Quantification of formation of pRPA2 S4/8 and RAD51 foci after 6 h CPT treatment in U2OS cells treated with indicated siRNA. pRPA2 S4/8 n = 5, RAD51 n =3 independent experiments ± S.E.M, compared to siNT. siNT, siRNA not-targeted. The statistical significance was determined by using two-tailed paired t test.
