RPA and RAD51 recruitment to stalled forks in FANCD2^−/−SLFN11^−/−HAP1 cells. (A) RPA and RAD51 foci formation in FANCD2^−/− and FANCD2^−/−SLFN11^−/− HAP1 cells treated or not with MMC (100 ng/mL, 24 hours). Means ± standard error of the mean (SEM) are shown. More than 300 nuclei were examined. Representative results from 2 independent experiments are shown. (B) RPA and RAD51 foci formation in FANCD2^−/− and FANCD2^−/−SLFN11^−/− HAP1 cells on HU treatment (4 mM, 5 hours). More than 300 nuclei were examined. Representative results from 2 independent experiments are shown. (C) PLA between the nascent DNA (IdU tract) and RAD51. Cells were transfected with control or siRNA targeting DNA2 and then 48 hours later, cells were pulsed for 30 minutes with IdU and then treated with HU (4 mM, 5 hours). PLA was performed in a native condition as described previously.³⁶  PLA performed with anti-IdU alone served as a negative control. Means ± SEM with representative images from 2 independent experiments are shown. P values were calculated by unpaired, 2-tailed Student t tests. A schematic illustration of the PLA assay is shown.