SLFN11 deficiency prevents replication fork degradation. (A) A schema of the experimental protocol. (a) Overview of the experiment. Cells are sequentially pulse-labeled by IdU and CldU to visualize nascent DNA tracts and further treated with HU or MMC. The shortened length of the CldU tracts indicates the degree of fork degradation. (b) The stalled forks are remodeled and reversed into a 4-way junction form, which is a target of the nucleases. (c) The DNA fiber protocol. Pulse-labeled cells were fixed, spotted on a slide glass, and lyzed. The slide glass is tilted to spread DNA fibers. Finally, the IdU and CldU tracts are detected by specific antibodies and visualized by an In Cell Analyzer. (B) DNA fiber analysis to quantify fork degradation/protection. CldU tract length was measured in PD20 cells in which SLFN11 expression was knocked down by siRNA transfection with or without HU treatment (4 mM, 5 hours). The tract lengths were quantified in 3 independent experiments, and the cumulative results of more than 450 fibers with means ± standard error of the mean (SEM) are shown. P values were calculated by unpaired, 2-tailed Student t tests. HAP1 cells with the indicated genotypes were analyzed for fork degradation/protection in the presence of HU (C) or MMC (D). (E) HAP1 FANCD2^−/−SLFN11^−/− cells complemented with wild-type or an ATPase-dead SLFN11 mutant were analyzed by the DNA fiber assay. (F) Quantification of restarting forks in HAP1 FANCD2^−/−SLFN11^−/− cells. The percentage of restarting forks was calculated in the cumulative results of more than 300 fibers from triplicate experiments. Means ± SEM are shown. P values were calculated by unpaired, 2-tailed Student t tests.