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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 3. — RFWD3 promotes the recruitment of ZRANB3 to ubiquitinated sites of DNA damage. (A) Localization of HA-ZRANB3 to DNA damage sites generated by UV laser microirradiation. U2OS cells expressing HA-ZRANB3 were transfected with the indicated siRNAs, fixed 30 min after laser irradiation, and stained with anti-HA (green) and anti-γH2AX (red) antibodies. Images are representative of results quantitated in Fig. 3 B. Scale bars, 10 µm. (B) Graph showing the percentage of γH2AX-positive U2OS cells with HA-ZRANB3 colocalization at UV laser stripes. Data correspond to conditions in Fig. 3 A and represent the mean and SD (**P < 0.01, unpaired t test). (C) Detection of RFWD3 and HA-ZRANB3 levels in U2OS cells used in Fig. 3, E and F. Immunoblot was performed with antibodies against RFWD3 and HA. (D) U2OS cells expressing HA-ZRANB3 were transfected with siUSP1 to induce ZRANB3 localization to nuclear foci in the absence of exogenous DNA damage. Cells were also co-transfected with the indicated control or RFWD3 siRNAs and stained with anti-HA antibody (green). Scale bars, 5 µm. (E) Percentage of U2OS cells with more than ten HA-ZRANB3 foci upon co-transfection of siUSP1 and the indicated siRNAs, as described in Fig. 3 D. Data represent the mean and SD from three independent experiments (**P < 0.01; ***P < 0.001; unpaired t test). (F) U2OS cells expressing HA-ZRANB3 were transfected as in Fig. 3 D. The mean intensity of HA-ZRANB3 foci in each cell is depicted as a box plot for a population of more than 200 cells for each siRNA condition. Whiskers represent the minimum and maximum (n.s., not significant; ****P < 0.0001; Mann Whitney test).