Fig. 3.

Effect of the zinc finger variants on cell proliferation and apoptosis. A Click-IT EdU proliferation assay was performed on fibroblasts from affected individuals 1 and 2, and 2 independent control fibroblast lines. Cells that did incorporate EdU during incubation are shown in red, all nuclei were counterstained with DAPI (blue). Scale bars indicate 20 µm. B Quantification of the percentage of proliferating fibroblasts. The number of EdU + (red) cells was calculated as a percentage of all DAPI + (blue) nuclei (One-way ANOVA, ****p < 0.0001, mean ± SEM, n =  > 8–10 fields per slide, n = 3 experiments). C Number of apoptotic and necrotic cells was determined with flow cytometry after staining with FAM–FLICA and propidium iodide (PI). Cells were sorted on apoptotic signal intensity (FAM–FLICA/caspase 3/7) and necrotic signal intensity (PI), which resulted in the formation of four clusters. Cluster Q1 depicts necrotic cells, Q2 shows late apoptotic cells, Q3 shows early apoptotic cells and Q4 shows healthy cells. D Number of apoptotic cells (Q2 + Q3) was calculated as a percentage of all cells analyzed (n = 3 experiments, n =  > 10.000 cells per analysis, One-way ANOVA: *p = 0.034, ***p = 0.0002, mean ± SEM)