Fig. 4: CX3 complex promotes reversed fork restart in U2OS cells.
From: Sequential role of RAD51 paralog complexes in replication fork remodeling and restart
a DNA fiber analysis of U2OS cells transfected for 24–48 h (24 h for RAD51, 48 h for all the other genes) with a control siRNA (Ctrl) or with siRNAs targeting RAD51, RAD51C, RAD51D, and XRCC3 to investigate replication fork restart upon HU. Top: schematic CldU/IdU pulse-labeling protocol to evaluate fork restart upon HU treatment (2 mM, 2 h). Bottom-left: representative stalled and restarting forks. Bottom-right: frequency of fork stalling for each genetic condition. In a–d, the graph-bar depicts mean and SD from three independent experiments. Statistical analysis: one-way ANOVA followed by Bonferroni test; ns not significant; ****p-value < 0.0001. b DNA fiber analysis of U2OS cells double transfected with a control siRNA (Ctrl) or with a siRNA targeting XRCC3 (48 h) or RAD51 (24 h) to investigate replication fork restart upon HU treatments; labeling protocol as in a. Top: levels of indicated proteins, assessed by western blot; Tubulin, loading control. In b and d multiple gels/blots were processed in parallel, ensuring equal and comparable loading across gels. Bottom: frequency of fork stalling. ns not significant; **p-value = 0.0068 (1 versus 3) and 0.0087 (3 versus 4). c DNA fiber analysis of wild-type (WT) or ZRANB3 Knock-Out (KO) U2OS cells transfected with a control siRNA (Ctrl) or with siRNAs targeting XRCC3 (48 h) to investigate replication fork restart upon HU treatments; labeling protocol as in a. Top: indicated protein levels, assessed by western blot; Tubulin, loading control; asterisk, specific band. Bottom: frequency of fork stalling. ns not significant; ****p-value < 0.001. d DNA fiber analysis of U2OS cells double transfected with a control siRNA (Ctrl), with siRNAs targeting XRCC3 (48 h) or RAD51D (60 h) to investigate replication fork restart upon HU treatments; labeling protocol as in a. Top: indicated protein levels, assessed by western blot; KU70, loading control. Bottom: frequency of fork stalling. ns not significant; **p-value = 0.0024; ***p-value = 0.0007. Source data for a–d are provided in the Source Data file.
