Fig. 4: SART3 depletion upregulates DSBs-induced DNA‒RNA hybrids.

a, b U2OS cells were transfected with siNC or siSART3. After 36 h, cells were treated with 5 µM CPT for 2 h, followed by S9.6 immunostaining. The numbers represent mean S9.6 intensity per nucleus measured by Cellprofiler (a). N = 281, 391, 364, 338, 301, 302, 308, 348, 330 correspond to the nine groups shown on the x-axis, based on three replicates. Error bars represent mean ± SD. P values were calculated using a two-tailed Mann–Whitney U test. Representative images are shown (b), with the areas highlighted by circles quantified for S9.6 intensity analysis. Scale bar, 10 µm. c Cartoon deciphers DR-IP (top). U2OS-ER-AsiSI cells were transfected with siNC or siSART3. After 48 h, cells were treated with 4-OHT followed by DRIP-qPCR analysis (bottom). d DRIP-qPCR analysis was performed as in (c) supplemented with RNase H during genome cleavage. The primers spanning the AsiSI-induced HR-1 break site and the actin exon region were used for qPCR. The yellow shaded area represents RNase H treatment. e R-ChIP-qPCR analysis of DNA‒RNA hybrids. Cartoon deciphers R-ChIP (top). U2OS-DR-GFP cells stably expressing V5-RNase H1-D210N mutant were transfected with siNC or siSART3. 24 h later, cells were infected with I-SceI lentivirus. After 36 h, cells were harvested followed by R-ChIP. The levels of DNA-RNA hybrids around DSBs were detected by qPCR. In (c–e), error bars represent mean ± SEM (N = 3 independent experiments), and p values were calculated using unpaired two-tailed Student’s t test. Source data are provided as a Source Data file.