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. 2020 Aug 31;130(10):5272–5286. doi: 10.1172/JCI134966

Figure 5. R426C/R582X mutations lead to increased nuclear area and γH2AX staining in immortalized fibroblasts.

Figure 5

(A) Immortalized fibroblasts were fixed, permeabilized, and incubated with primary anti-MCM10 antibody followed by goat anti-mouse Alexa Fluor 488 secondary antibody and directly conjugated anti-γH2AX Alexa Fluor 647. Slides were mounted with ProLong Gold antifade media with DAPI and imaged by confocal microscopy. Scale bars: 10 μm. (B) MFI of γH2AX staining (left) and area of positive γH2AX signal (right) were measured in 28 to 35 cells per condition. ****P < 0.0001, unpaired t test (left) or Mann-Whitney U test (right). Data are represented as mean ± 95% CI. (C) Nuclear area was measured by positive DAPI staining in 31 (patient) and 46 (healthy donor) cells per condition. ****P < 0.0001, Mann-Whitney U test. Data are represented as mean ± 95% CI. (D) R426C or R582X variants were transiently overexpressed in 293T cells and prepared for microscopy, as described above. Nuclear area determined by DAPI staining was measured. *P < 0.05, Kruskal-Wallis with Dunn’s multiple comparison test. Data are represented as mean ± 95% CI. n = 69 (R426C); n = 95 (R582X, WT). (E) Healthy donor–derived (left) or patient-derived (right) immortalized fibroblast cells were labeled with BRDU and 7-AAD, and cell cycle was analyzed by FACS. All data shown are of 1 representative experiment of 3 technical replicates performed on different days.