Rag1−/− zebrafish present with altered survival and cellular features, potentially indicating severe DNA damage. (a) Kaplan–Meier representation of the survival of rag1−/− zebrafish and their wt siblings (n = 72 rag1−/−, n = 128 wt). The log‐rank (Mantel–Cox) test was used for statistical analysis (p < 0.0001). (b) Cell cycle distribution of kidney cells based on the amount of DNA present. Cells were isolated from wt and rag1−/− fish, stained with propidium iodide, and examined by flow cytometry. Representative histograms from one wt and rag1−/− fish are shown. Apoptotic cells are clearly visible in the rag1−/− fish. (c) The percentage of cells in G0/G1, S, and G2/M was analyzed using ModFit LT 4.1 software. The stacked graphs represent the means ± SEM of the fish from the different groups. Significant differences in the percentage of G1 and G2/M cells were observed between wt and rag1−/− at the age of 1 year and 9 months (*p < 0.05). (d) Percentage of apoptotic cells in the kidneys of wt and rag1−/− fish, as determined by TUNEL assays. Positive cells were counted in 20 images. The means ± SEM are presented. (e) The dynamics of telomeres in rag1−/− zebrafish. Telomere length of 8‐ and 12‐month‐old zebrafish with the indicated genetic backgrounds, as assayed by FLOW‐FISH. The results are shown as the means ± SEM of four fish; p < 0.05 according to Student's t test. a.u.f, arbitrary units of fluorescence. (f) Immunocytochemical quantification of histone γ‐H2AX in kidney cells from wt and rag1−/− fish (aged 1 year and 9 months old). The number of positive cells was counted in 35 images. The means ± SEM are presented; p < 0.001 significantly different from wt fish. Confocal images of kidney cells reveal the location of histone γ‐H2AX foci in wt and rag1−/− fish. Nuclei are stained with DAPI and are shown as blue. Histone γ‐H2AX was observed using the Alexa Fluor 568 secondary antibody and is presented as pink. Scale bar = 25 µm. In all experiments, the results represent the means ± SEM of the percentage of positive cells. Mann–Whitney U tests were conducted to compare the means using GraphPad Prism 6 software (p < 0.001)
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