Figure 6.

RFWD3 stimulates ZRANB3 recruitment and replication fork reversal in BRCA2-deficient cells. (A) Immunoblot showing RFWD3, BRCA2, and HA-ZRANB3 levels in U2OS cells used in Fig. 6, B–E. HA-ZRANB3 was detected with anti-HA antibody. (B) U2OS cells expressing HA-ZRANB3 were transfected with siRFWD3-2 and/or siBRCA2-3, fixed 30 min after UV laser irradiation, and stained with anti-HA (green) and anti-γH2AX (red) antibodies. Scale bars, 10 µm. (C) Graph showing the percentage of γH2AX-positive cells with HA-ZRANB3 colocalization at UV laser stripes corresponding to the experiment in Fig. 6 B. Data represent the mean and SD from two independent experiments (**P < 0.01, unpaired t test). (D) U2OS cells expressing HA-ZRANB3 were transfected with siRFWD3-2 and/or siBRCA2-3 and treated with 10 µM EdU for 10 min followed by 1 µg/ml 4NQO for 4 h. After cell fixation, biotin was conjugated to EdU by click chemistry, and proximity ligation assay (PLA) was performed with anti-HA and anti-biotin antibodies. Images are representative of results quantitated in Fig. 6 E. Scale bars, 5 µm. (E) Box plot showing the distribution of PLA foci per cell for each condition (>200 cells) in Fig. 6 D. Whiskers represent the 10th and 90th percentiles (****P < 0.0001, Mann Whitney test). (F) Detection of RFWD3 and BRCA2 levels in U2OS cells used in Fig. 6 G. (G) U2OS cells were transfected with siRFWD3-4 and/or siBRCA2-3 and treated with 2 mM HU for 5 h. Where indicated, 50 µM mirin was added concurrently with HU. 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.