Fig. 5. The ratio of differentiating cells to proliferative cells correlates with the level of Hdac activity. (A) Western blot analysis of 24 hpf wild-type and add mutant embryos using anti-acetylated histone H4 antibody. (B) Treatment of wild-type embryos with 1200 nM TSA from 14 hpf. (C) Treatment of wild-type embryos with 400 nM TSA from 14 hpf. (D-G) BrdU labeling of 2 dpf wild-type embryos treated with TSA from 14 hpf at different concentrations: 1200 nM (D), 800 nM (E), 400 nM (F) and control DMSO (G). Asterisk (E) indicates BrdU-negative area in presumptive retinal ganglion cell layer. (H) Western blot analysis of 24 hpf wild-type embryos treated with TSA from 14 hpf at different concentrations using antibodies against acetylated histone H4 (upper panel) and total histone H4 (lower panel). (I) Histogram of percentage of the BrdU-positive retinal cells (left, red bars) and histone acetylation level (right, blue bars). The percentage of BrdU-positive cells in 1200 nM TSA treatment is underestimated because of massive cell death (asterisk). RGC, retinal ganglion cell layer.