Fig. 3: BCDX2 complex, but not CX3, promotes reversed fork degradation in BRCA2-depleted U2OS cells.

a, b DNA fiber analysis of U2OS cells double transfected with a control siRNA (Ctrl) or with siRNAs targeting RAD51C, RAD51D, and XRCC3 for 60 h and with BRCA2 siRNA for 48 h. a Top: schematic CldU/IdU pulse-labeling protocol to evaluate fork degradation upon HU treatment (4 mM, 5 h). Bottom: representative DNA fibers from each genetic condition. b IdU/CIdU ratio is plotted as a readout of fork degradation. In b and c, the numbers indicate the mean value; a minimum of 150 forks was scored in two independent experiments yielding similar results. Bounds of box are 25–75th percentile, center shows the median, whiskers indicate the 10–90 percentiles, data outside this range are drawn as individual dots. Statistical analysis: Kruskal–Wallis test; ns not significant; ****p-value < 0.0001. c DNA fiber analysis of KO and reconstituted (+) U2OS cells transfected with a control siRNA (Ctrl) or with BRCA2 siRNA for 48 h and consecutively labeled and HU-treated as in a. IdU/CIdU ratio is plotted as a readout of fork degradation. d Frequency of reversed replication forks in U2OS cells transfected with control siRNA (Ctrl) or with siRNAs targeting BRCA2, RAD51C, or XRCC3 for 48 h and treated for 5 h with HU 4 mM; where indicated 50 μM Mirin was added 1 h before HU treatment (6 h total treatment). Graph-bar depicts mean and SD from three independent EM experiments. In brackets, total number of molecules analyzed per condition. Statistical analysis: one-way ANOVA followed by Bonferroni test; ns not significant; ***p-value = 0.0003. Source data for a–d are provided in the Source Data file.