Fig. 5

9-HSA Inhibits HDAC1 Enzymatic Activity In Vivo in the Retina

(A–H) 9-HSA-treated and hdac^−/− mutant retinae showed an increase in histone H4 hyperacetylation marks in comparison to wild-type (hdac1^+/+) and DMSO-treated retinae at 2 and 3 days postfertilization (dpf). Immunohistochemistry of anti-hyperacetylated histone H4 (acH4, green) on 2 and 3 dpf frontal retinal cryosections.

(I) Western blot analysis of histone H4 and histone H3 hyperacetylation marks (acH4 and acH3K9 respectively) from 3 dpf DMSO-treated, wild-type (hdac1^+/+), 9-HSA-treated, and hdac1^−/− whole embryo lysates showed an increase in both marks in 9-HSA-treated and hdac1^−/− whole embryo lysates.

(J) Western blot quantification of hyperacetylation of the histone 4 (acH4) and histone 3 (acH3K9) marks. Data are presented as ratios of 9-HSA-treated over DMSO-treated embryos and hdac1^−/− mutant over wild-type (hdac1^+/+). Values are means ± standard error of three independent samples.

(K) Quantitative real-time PCR of hdac1 expression level in 2 dpf 9-HSA, DMSO-injected embryos as well as hdac1^−/− and wild-type sibling (hdac1^+/+) embryos was performed. No difference could be detected in the level of expression of hdac1 between both conditions in contrast to the expected loss of hdac1 expression in the hdac1 mutant (n = 3 for all conditions; p value = 0.9216 for 9-HSA/DMSO, p value < 0.0001 for hdac1 mutant/wild-type sibling. Statistical significance was determined using a Student’s t test and depicted as: ^∗p value < 0.05; ns = nonsignificant. Scale bars, 20 μm in (A)–(D) and 50 μm (E)–(H).