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

Figure 2.

Figure 2.

Mutations in the RFWD3 ubiquitin ligase and WD40 domains rescue nascent DNA degradation in BRCA2-deficient cells. (A) Detection of RFWD3 and BRCA2 levels in U2OS cells for the experiment in Fig. 2 B. (B) U2OS cells expressing siRNA-resistant RFWD3 (WT or C315A) were transfected with siBRCA2-3 and siRFWD3-4, and they were compared with U2OS cells transfected with siBRCA2-3 with or without siRFWD3-4. Cells 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) Detection of RFWD3 and BRCA2 levels in FA patient fibroblasts for the experiment in Fig. 2 D. (D) Schematic for single DNA fiber analysis to detect nascent DNA degradation at stalled forks in FA patient fibroblasts complemented with WT RFWD3 (1143 + WT) or empty vector (1143 + mock). Cells were labeled with sequential CldU (40 min) and IdU (50 min) and then treated with 2 mM HU (5 h). Representative images are provided for CldU and IdU-containing replication tracks upon transfection with siFF or siBRCA2-3. Scale bars, 5 µm. (E) IdU/CldU replication length ratios in FA patient fibroblasts treated as in Fig. 2 D. Median values from >200 replication tracks are represented by red lines (n.s., not significant; ****P < 0.0001; Mann Whitney test).