Figure 5.

H3.3 Knockdown decreases the acetylation of H4K16. (a,b) Protein levels of different transcriptional activation markers are detected in the H3.3 knockdown NSCs versus the control. Among them, H4K16 is selectively downregulated (n=3 independent experiments; bar represents mean±S.E.M; **P<0.01; Lamin A/C served as loading control). (c,d) H4K16ac, H3.3, are detected at various time points (E10, E13, E15, E17, and P0) in cortical lysates. Samples for western blot analysis are shown. A scatter diagram shows the change trend of H4K16ac, H3.3, and different neurogenesis markers (n=3 independent experiments; bar represents mean±S.E.M; β-ACTIN served as loading control). (e) NSCs were isolated from the E12.5 mice brains and cultured in the proliferative medium for 24 h. The cells were co-stained with anti-H4K16ac and anti-H3.3 antibodies; anti-H4K16ac anti-SOX2 antibodies; anti-H4K16ac and anti-NESTIN antibodies. Scale bar represents 25μm. (f,g) Protein level change of H4K16ac can be rescued by H3.3 overexpression in NSCs (n=3 independent experiments; bar represents mean±S.E.M; **P<0.01; Lamin A/C served as loading control). (h,i) The level of H4K16ac was gradually reduced by gradually increased dose of H3.3KD#2 shRNA-mediated interference in NSC cells (n=3 independent experiments; bar represents mean±S.E.M; **P<0.01; Lamin A/C served as loading control)