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. 2023 Apr 10;222(5):e202106022. doi: 10.1083/jcb.202106022

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© 2023 Moore et al.

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Figure S2. — Effect of RFWD3 depletion on ZRANB3 and PCNA recruitment. (A) U2OS cells expressing HA-ZRANB3 were transfected with the indicated siRNAs, fixed 10 min after UV laser irradiation, and stained with anti-HA and anti-γH2AX antibodies. The percentage of γH2AX-positive cells with HA-ZRANB3 colocalization at laser-generated stripes was quantitated. Data represent the mean and SD from three independent experiments (n.s., not significant; unpaired t test). (B) U2OS cells expressing HA-ZRANB3 were transfected with siUSP1 to induce ZRANB3 localization to nuclear foci in the absence of exogenous DNA damage. Foci were detected by staining with anti-HA antibody (green). Immunoblot shows the level of USP1 depletion. Scale bars, 10 µm. (C) Percentage of U2OS cells with more than ten HA-ZRANB3 foci upon transfection of siUSP1 as described in Fig. S2 B. Data represent the mean and SD from two independent experiments (***P < 0.001; unpaired t test). (D) U2OS cells expressing HA-ZRANB3 were treated as in Fig. 4 B except PLA was performed with either anti-HA or anti-biotin antibody alone as negative controls. All conditions were treated with 4NQO. Whiskers on box plots represent the 10th and 90th percentiles for >200 cells. (E) U2OS cells expressing HA-ZRANB3 were transduced with shRFWD3-4 or non-targeting shFF along with either shRNA-resistant WT or C315A RFWD3. They were pulsed with 10 µM EdU for 10 min, treated with 4 mM HU and 10 µM EdU for 4 h, and fixed. Biotin was conjugated to EdU by click chemistry, and PLA was performed with anti-HA and anti-biotin antibodies. Whiskers on box plots (>200 cells) represent the 10th and 90th percentiles (n.s., not significant; ****P < 0.0001; Mann Whitney test). (F) U2OS cells expressing HA-ZRANB3 were treated as in Fig. 4, D–F except PLA was performed with anti-PCNA and anti-biotin antibodies. Whiskers on box plots (>200 cells) represent the 10th and 90th percentiles (n.s., not significant; Mann Whitney test). RFWD3 depletion efficiency is provided in Fig. 4 D. (G) U2OS cells expressing HA-ZRANB3 were transduced with the indicated shRNAs, pulsed with 15 µM EdU for 10 min, chased with 15 µM thymidine for 2 h, treated with 1 µg/ml 4NQO for 4 h, and fixed. Biotin was conjugated to EdU, and PLA was performed with anti-HA and anti-biotin antibodies. Whiskers on box plots (>200 cells) represent the 10th and 90th percentiles (n.s., not significant; Mann Whitney test). Immunoblot shows level of RFWD3 depletion.