Figure 4: The LEMD2 p.L13R Mutation Inhibits Proliferation, Induces Cell Senescence, and Cell Cycle Arrest in Fibroblasts

(A) Cell proliferation assay based on confluence from passage 2 (P2) in the left panel and P15 in the right panel in patient (Pat) fibroblasts, Ctrl1, and an older age (“high-senescence”) control (Ctrl3). There was a significant difference in the rate of proliferation detected between both control subjects and patient at P2 and P15. The cells were imaged every 2 h for 90 h. n = 8 wells, mean ± SD, ***p < 0.001. (B) Images of β-galactosidase (β gal)-stained fibroblasts from patient and Ctrl1 as well as Ctrl3 at passage 6 (P6) and P15 in the left panel. Note that the amount of blue-stained cells is visibly higher in patient cells. (Right) Quantification of β gal–stained cells revealed increased cell senescence in patient cells compared with both control subjects at passages as indicated (n = 3; **p < 0.01; ***p < 0.001). (C) Fibroblasts stained with propidium iodide and measurements of the deoxyribonucleic acid content for each phase of the cell cycle by flow cytometry. Cells were taken from Ctrl1 at P6 and 9, from the patient (Pat) at passage 8 and passage 11, and from Ctrl3 at passage 12. Representative diagrams of each phase of the cell cycle are shown in the left panel. (Right) Quantification of the deoxyribonucleic acid content showed a potential arrest in the G1 phase in patient fibroblasts compared with Ctrl1 and similar to Ctrl3 at later passage. n = 3; ∗∗p < 0.01; ∗p < 0.05; ***p < 0.001. (D) Western blot of Aurora B protein expression in patient and control cells on the left panel. GAPDH was used as loading control. (Right) Quantification by densitometry of Aurora B protein expression revealed a significant difference between both control subjects and patient fibroblasts. n = 3 experiments with each 2 to 3 replicates; ***p < 0.001. Abbreviations as in Figures 2 and 3.