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. 2024 Oct 5;15:8648. doi: 10.1038/s41467-024-52916-z

Fig. 6. SMARCAD1 displaces 53BP1 at γH2AX sites on broken forks to promote BIR.

Fig. 6

a Recruitment of RAD51 (left and middle) and 53BP1 (left and right) to γH2AX sites was analyzed by PLA in U2OS cells treated with IR (4 Gy, 2 h after for PLA) or HU (1 mM, 24 h, 30 min after release for PLA). Left: representative PLA images. Right: quantification of PLA foci per nucleus. Also see Supplementary Fig. 9a. Scale bar = 2 μm. (n = 300 cells). b Recruitment of 53BP1 (left), PCNA (middle) and PIF1 (right) to γH2AX sites was analyzed by PLA in U2OS cells expressing SMARCAD1 shRNAs with vector as a control after HU treatment (1 mM, 24 h). Quantification of PLA foci per nucleus is displayed. Also see Supplementary Fig. 9b. (n = 300 cells). c Recruitment of PCNA (left) and PIF1 (right) to γH2AX sites was analyzed by PLA in U2OS WT and 53BP1-KO cells after HU treatment (1 mM, 24 h). Quantification of PLA foci per nucleus is displayed. Also see Supplementary Fig. 9c. (n = 300 cells). d, e Recruitment of 53BP1 (d), PCNA (e, left) and PIF1 (e, right) to γH2AX sites was analyzed by PLA in U2OS cells expressing Flag-SMACRAD1-WT or Flag-NΔ-SMACRAD1 with endogenous SMACRAD1 depleted by shRNA after HU treatment (1 mM, 24 h). Quantification of PLA foci per nucleus is displayed. Also see Supplementary Fig. 9d for d and Supplementary Fig. 9e for e. (n = 300 cells). a to e: Three experiments were performed with ∼100 nuclei analyzed in each experiment. Quantification of PLA foci per nucleus from a total of ~300 nuclei are displayed. f HR frequency was determined in U2OS (EGFP-HR/STGC) cells expressing SMACRAD1 shRNA with a vector control (left) or in cells expressing Flag-SMACRAD1-WT or Flag-NΔ-SMACRAD1 with endogenous SMACRAD1 depleted by shRNAs, 5 days after I-Sce1 lentiviral infection. Also see Supplementary Fig. 9d. (n = 5 replicates). g U2OS (EGFP-BIR/LTGC-Flex1) cells expressing Flag-SMACRAD1-WT or Flag-NΔ-SMACRAD1 with endogenous SMACRAD1 depleted by shRNA were further infected with lentiviruses encoding FANCM shRNA (left) or synchronized to S-phase using double thymidine block followed by HU treatment (1 mM, 24 h, right). BIR frequency was assessed by FACS 6 days after. Also see S9f. (n = 5 replicates). Source data are provided as a Source data file.