Fig. 4: RNF25 protects reversed replication forks from degradation.

a H1299 WT and two independent RNF25 KO clones were treated with 2 mM HU for 5 h, then washed and nuclease-released chromatin fraction timepoints taken at 0, 1, and 2 h after release from HU for analysis by immunoblotting. Nuclease-released chromatin fractions from cells not treated with HU (lanes 1-3) were also collected. b Top: Schematic of fork degradation assays. Bottom: Pa02C and Pa03C sgNT and sgRNF25 cells were labeled with CldU and IdU, then treated with HU to induce fork stalling. The ratio of IdU to CldU tracts was used as a readout of fork degradation. c Fork degradation assay in H1299 WT cells and two independent RNF25 KO clones, as well as H1299 WT cells treated with siControl, and two independent siRNAs targeting RNF25. d H1299 WT and RNF25 KO cells were transfected with indicated siRNAs for 48 h before conducting the fork degradation assay. e SIRF assay between HA-RNF25 and EdU in H1299 RNF25 KO cells. Left: representative images of foci formation; scale bar is 10 μm. Right: the number of foci per nucleus was quantified using ImageJ. Each biological replicate is shown in blue and pink, with means indicated with bars. Number of nuclei quantified per condition (replicate 1-blue, replicate 2-pink): EV: (84, 79); HA-RNF25 WT: (90, 75). Statistics: two-tailed Student’s t test on Log₂-transformed data. All DNA fiber assays shown (b-d) are a representative experiment from two biological replicates. A total of 150 fibers were measured per condition and statistics analyzed using a two-tailed Mann-Whitney test. Means are indicated with bars. Source data are provided as a Source Data file.