Figure 4. Biallelic JMJD5 pathogenic variants are associated with increased markers of replication stress.

Immortalized fibroblasts were monitored for 2 commonly used markers of replication stress (micronuclei and 53BP1 bodies). The affected Sib4_In/CY fibroblasts show significantly increased replication stress. (A) 53BP1 bodies were counted using immunofluorescence staining and distinguished from 53BP1 foci based on their size and presence only in G₁ cells (using costaining for CENPF). Shown are examples of cells with different numbers of 53BP1 bodies. (B) 53BP1 bodies in untreated cells were significantly increased in Sib4_In/CY immortalized fibroblasts. APH indicates aphidicolin, a DNA polymerase inhibitor that is an established replication stress stimulus (see below). (C) 53BP1 bodies were also significantly increased in Sib4_In/CY immortalized fibroblasts treated with APH (0.5 μM for 48 hours). (D) Micronuclei were counted following DAPI staining. Shown are 2 examples of cells with micronuclei. (E) Micronuclei were significantly increased in untreated Sib4_In/CY immortalized fibroblasts. (F) Micronuclei were also significantly increased in Sib4_In/CY immortalized fibroblasts treated with APH. (B, C, E, and F) Data represent mean ± SEM from 5 independent experiments. For 53BP1 bodies, a minimum of 300 cells were counted per sample. For micronuclei, a minimum of 500 cells were counted per sample. Statistical analyses used 1-way ANOVA with Tukey’s post hoc test; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.