FIGURE 7.

Activity-dependent phosphorylation of histone H3 at serine 10 requires afadin nuclear translocation. A, representative high magnification images of cortical neurons costained for H3S10p and afadin following activity-dependent stimulation. Red dashed lines outline the nucleus (DAPI) in lower panels. B, quantification of H3S10p in afadin-positive cells revealed an increase in H3 phosphorylation 30, 120, and 240 min post-stimulation; neurons also display an increase in afadin nuclear content 120 min post-treatment (*, p < 0.05, ANOVA). C, representative high magnification images of neurons overexpressing, or not, Myc-afadin-NT and costained for H3S10p, following NMDA receptor activation. Red dashed lines outline the nucleus (DAPI) in lower panels. D, quantification of H3S10p in C revealed that in cells expressing Myc-afadin-NT H3 phosphorylation levels are significantly decreased compared with nonexpressing cells (*, p < 0.05; ANOVA). E, low magnification images of H3S10p and Myc-stained cortical neurons following activity-dependent stimulation for 0, 30, 120, or 240 min. Cortical neurons (DIV 25) were transfected with Myc-afadin-NT or not and probed with H3S10p. Red dashed boxes indicate cells expressing Myc-afadin-NT, and yellow dashed boxes enclose cells not expressing Myc-afadin-NT. H3S10p (images are pseudo-colored) increases in a time-dependent manner after activity-dependent stimulation in non-Myc-afadin-NT expressing cells. In Myc-afadin-NT expressing cells, H3S10p levels are significantly increased after 30 min but not at 120 or 240 min. F, quantification of relative H3S10p intensity in stimulated nontransfected and Myc-afadin-NT expressing cells shown in yellow boxes (nontransfected) or red boxes (transfected) in E. (**, p < 0.01; ***, p < 0.001, two-way ANOVA.) Scale bar, 15 μm. APVwd, activity-dependent stimulation.