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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 7. — RFWD3 and ZRANB3 epistasis in replication fork remodeling phenotypes. (A) Detection of RFWD3, ZRANB3, and BRCA2 levels in U2OS cells used in Fig. 7, B and C. (B) U2OS cells were transfected with siBRCA2-3, siRFWD3-4, and/or siZRANB3. They were labeled with sequential CldU (25 min) and IdU (30 min) and then treated with 2 mM HU (5 h) as in Fig. 1 C. Median values for IdU/CldU track ratios (from >200 tracks) are represented by red lines (n.s., not significant; ****P < 0.0001; Mann Whitney test). (C) Neutral comet-tail moments in U2OS cells transfected with siBRCA2-3, siRFWD3-4, and/or siZRANB3 and treated with 2 mM HU for 24 h. Whiskers represent the 10th and 90th percentiles. More than 200 cells were analyzed for each condition (****P < 0.0001, Mann Whitney test). (D) Detection of RFWD3 and ZRANB3 levels in U2OS cells used in Fig. 7 E. (E) U2OS cells transfected with siRFWD3-4 and/or siZRANB3 were treated with 2 mM HU for 5 h. Replication intermediates were detected by electron microscopy, and the percentage of reversed forks was measured in a single replicate. The number of replication intermediates analyzed for each condition is indicated in parentheses.