Figure 5. Biallelic JMJD5 pathogenic variants are associated with impaired DNA replication fidelity and reduced colony survival.

(A) DNA fiber assays were performed by incubation of cells with thymidine analog CldU followed by IdU, with detection by immunofluorescence (top). Examples of different labeling outcomes are shown (bottom). (B) Stalled replication forks were significantly increased in Sib4_In/CY immortalized fibroblasts. The prevalence of stalled forks was calculated as a percentage of total fork structures counted. A minimum of 200 forks were counted per condition in each experiment. (C) Asymmetric replication forks were significantly increased in Sib4_In/CY immortalized fibroblasts. Fork symmetry was determined by measurement of the ratio between the two IdU tracts found in first-label origin structures. Symmetric forks should approach a ratio of about 1.0. An increase indicates fork asymmetry. At least 50 structures were measured per sample in each experiment. (D) The total length of ongoing DNA fiber fork structures was measured and converted to replication fork speed. No significant difference in replication fork speed was observed. A minimum of 200 forks were analyzed per sample. (E) Fibroblasts were plated at limited density and monitored for colony formation. Sib4_In/CY fibroblasts had reduced colony formation. (B–D) Data represent mean ± SEM from 4 independent experiments. (E) Data represent mean ± SEM from 3 independent experiments. Statistical analyses used 1-way ANOVA with Tukey’s post hoc test (B and E) or Kruskal-Wallis with Dunn’s correction (C and D); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.