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. 2021 Jan 11;118(3):e2010370118. doi: 10.1073/pnas.2010370118

Fig. 1.

Fig. 1.

ATRX and RECQ5 define distinct subpathways of HR. (A) U2OSATRX cells, with and without doxycycline-induced ATRX expression, were transfected with siCtrl or siRECQ5 and treated with RAD51 inhibitor (RAD51i) prior to IR and throughout repair incubation. γH2AX foci were enumerated in EdU-negative G2 cells. Spontaneous foci (four to six) were subtracted. Knockdown and ATRX expression were confirmed by immunoblotting. Representative images of γH2AX foci and ATRX expression are shown. (B) U2OS cells were transfected with siCtrl or siRECQ5-2 and GFP, GFP-RECQ5-WT, GFP-RECQ5-ATP, or GFP-RECQ5-PIP, irradiated and γH2AX foci were enumerated in GFP-positive, EdU-negative G2 cells. Knockdown of the endogenous RECQ5 and GFP-RECQ5 expression levels were confirmed by immunoblotting (SI Appendix, Fig. S2A). Spontaneous foci (four to six) were subtracted. Representative images of γH2AX foci in GFP-positive cells are shown. (C) U2OS cells were transfected with GFP, GFP-ATRX-WT, GFP-ATRX-ATP, or GFP-ATRX-PIP, irradiated and γH2AX foci were enumerated in GFP-positive, EdU-negative G2 cells. Spontaneous foci (four to six) were subtracted. All data show mean ± SEM (n = 3). Results from individual experiments, each derived from 40 cells, are indicated. *P < 0.05; **P < 0.01; ***P < 0.001; ns: not significant (two-tailed t test).