FIGURE 7

MPH attenuates H2AX phosphorylation induced by ppp2r2c knockdown in mice primary neural cells. (a) Experimental design for MPH treatment of ppp2r2c knockdown in isolated primary neural cells from newborn mice. (b–d) Representative confocal images and relevant quantification of γH2AX foci (magenta) in primary neurons (stained with β‐tubulin, green), neural progenitor cells (NPCs, stained with SOX2, green), and glial cells (stained with GFAP, green) that were dissected from new born mice and cultured for 1 week and then were transfected with siRNA and treated with or without MPH for 3 days (scale bars, 15 μm). White boxes indicate the enlarged regions (n = 3 independent experiments for neurons, n = 2 independent experiments for NPCs and glial cells, above five fields containing at least 100 cells were analyzed per condition; two‐way ANOVA). (e–g) Representative microscopy images and its quantification of SA‐β‐gal signals in primary neurons (stained with β‐tubulin, green), NPCs (stained with SOX2, green), and glial (stained with GFAP, green) cells (scale bars, 25 μm for neuron, 50 μm for NPCs and glial cells). Quantification analysis of percentage of SA‐β‐gal‐positive cells (n = 3 independent experiments for neurons, n = 2 independent experiments for NPCs and glial cells, more than 100 cells were analyzed per condition; two‐way ANOVA). (h) Relative mRNA expression levels of Ppp2r2c, Mcm2, Mcm5, Mcm6, and Top2a in primary mouse neurons, NPCs, and glial cells determined by RT‐qPCR (n = 5 independent experiments for neurons and NPCs; n = 3 independent experiments for glial cells; unpaired two‐sided t test). Data are means ± SEM. *p < 0.05, **p < 0.01