FIGURE 6

PP2A activators decrease the rate of neural cells with senescence markers in old fish. (a) Representative confocal image of NeuN (green) and γH2AX (magenta) co‐staining in the brain of 22‐month‐old fish treated with or without PP2A activators for 3 days (scale bars, 5 μm). (b) Quantification of γH2AX‐positive nuclei (positive values indicate at least five γH2AX foci) in (a) (n = 4 for 6 and 22 m; n = 7 for 22m‐MPH, n = 3 for 22m‐DT‐061 and 22m‐FTY720, over 100 nuclei were analyzed per fish; * represents statistical difference in NeuN+ group, ^# represents statistical difference in NeuN− group; one‐way ANOVA). (c) Relative mRNA expression levels of cdkn1a, cdkn2a/b, and key SASP components determined by RT‐qPCR in 6 and 22 m WT brains with or without MPH or DT‐061 treatment (n = 3 independent biological samples, every sample pool two brains; one‐way ANOVA). (d) Representative image of confocal section of NeuN (green) co‐staining with cdkn2a/b (magenta) and cdkn1a (gray) mRNA by RNA‐Scope in the OT of 6 and 22 m WT treated with or without MPH for 3 days (scale bars, 5 μm). Arrows and triangles point to the cdkn2a/b and cdkn1a mRNA signals, respectively. Quantification of the percentage of cdkn2a/b, cdkn1a positive cells, respectively (positive values indicate at least one mRNA signal) and percentage of double positive cells in neuronal (NeuN+) and non‐neuronal (NeuN−) cells (n = 5 for each group and over 100 nuclei were analyzed per fish, * represents statistical difference in NeuN+ group, ^# represents statistical difference in NeuN− group; one‐way ANOVA). (e) Representative images of confocal section of NeuN (green) co‐staining with tnfa (magenta) and il‐8 (gray) mRNA by RNA‐Scope in the OT of 6 and 22 m WT treated with or without MPH for 3 days (scale bars, 5 μm). Arrows and triangles point to the tnfa and il‐8 mRNA signals, respectively. Quantification of the percentage of tnfa, il‐8‐positive cells, respectively (positive values indicate at least one mRNA signal) and percentage of double positive cells in neuronal (NeuN+) and non‐neuronal (NeuN−) cells (n = 5 for each group and over 100 nuclei were analyzed per fish; * represents statistical difference in NeuN+ group, ^# represents statistical difference in NeuN− group; one‐way ANOVA). Data are shown in means ± SEM. *p < 0.05, **p < 0.01, ^# p < 0.05, ^## p < 0.01