Fig. 6: RNF25 and REV7 function in a common DNA replication fork protection pathway. | Nature Communications

Fig. 6: RNF25 and REV7 function in a common DNA replication fork protection pathway.

From: The RING finger E3 ligase RNF25 protects DNA replication forks independently of its canonical roles in ubiquitin signaling

Fig. 6

H1299 WT and RNF25 KO cells were transfected with indicated siRNAs 48 h before conducting the DNA fork movement assay (a) and the fork degradation assay (b). 150 fibers were analyzed per condition with means indicated with bars. Each Fig. shown is a representative experiment from two biological replicates. Statistics: two-tailed Mann-Whitney test. c HA-RNF25 WT and domain mutants were expressed in H1299 cells using adenoviral vectors, then immunoprecipitated with anti-HA beads. d SIRF assay between HA-RNF25 (WT and C135/138S) and EdU in H1299 RNF25 KO cells transfected with control or REV7 siRNA. The number of foci per nucleus was quantified using ImageJ. Each biological replicate is shown in purple and blue, with means indicated using bars. Number of nuclei quantified per condition (from left to right) (replicate 1-purple, replicate 2-blue): (99, 64); (75, 96); (77, 75); (84, 74); (76, 82); (78, 61); (88, 86); (87, 80); (75, 70); (81, 66); (76, 77); (77, 63). Statistics: one-way ANOVA with Tukey’s multiple comparisons test on Log2-transformed data. e SIRF assay between HA-REV7 (WT and ΔSB) and EdU in H1299 WT and RNF25 KO cells. The number of foci per nucleus was quantified using ImageJ. Each biological replicate is shown in blue and pink with means indicated using bars. Number of nuclei quantified per condition (from left to right) (replicate 1-blue, replicate 2-pink): (70, 75); (74, 77); (71, 87); (70, 76); (73, 85); (72, 76); (70, 76); (75, 86); (72, 83); (74, 75); (65, 80); (72, 79). Statistics: one-way ANOVA with Tukey’s multiple comparisons test on Log2-transformed data. f Heatmap depicting relative mRNA expression of replication and DNA damage repair factors in PDACs from TCGA. Samples were clustered as normal or tumor samples, with additional classification of tumor samples by mutation status of key PDAC driver genes and genome maintenance related features (RS – Replication Stress signature, HRD – Homologous Recombination Deficiency score, CA20 – Centrosome Amplification signature). Source data are provided as a Source Data file.

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