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. 2018 Aug 22;44(3):514–525. doi: 10.1038/s41386-018-0181-y

Fig. 4.

Fig. 4

Preadministration of RV rescues pCREB and Arc expression; enhanced H3K9me2 and G9a in the Rac1 gene promoter suppresses Rac1 expression and is rescued by preadministration of RV or SR in the adult mouse hippocampus caused by ethanol exposure in P7 mice. Mice were exposed to saline or high dose of ethanol for 8 h after administration (30 min) with or without RV (2.5 mg/kg), and pCREB (ai) and Arc (aii) protein levels were determined in HP brain samples by a western blot analysis. HP tissue extracts from adult mice exposed to saline or ethanol were subjected for western blot analysis to determine Rac1 expression (bi). The protein samples were equally loaded, confirmed with Ponceau S staining, and normalized with total proteins (CREB) followed by β-actin. Rac1 mRNA levels (bii) were determined in samples exposed to saline and ethanol (8 h) by qPCR analysis. Hprt mRNA was used as the internal control for normalization of Rac1 mRNA. Epigenetic analysis at the promoter region of the Rac1 gene (c–e). ChIP analysis of the Rac1 gene promoter in HP tissues from the saline and ethanol groups with anti-H3K9me2, anti-H3K14ac, anti-H4K8ac, anti-G9a, anti-HDAC1, or ant-CBP antibodies (c). HP tissues from saline and ethanol groups preadministered (30 min before) with or without RV (d) or SR (e) were immunoprecipitated with anti-H3K9me2 or anti-G9a antibodies. Levels of Rac1 gene promoter chromatin enrichment in the IPs were measured by RT-qPCR. IgG was used as negative control. Enrichment values were normalized to input values and represented as percentage of input. Mice were exposed to a high dose of ethanol for 8 h after preadministration (30 min) with vehicle or RV (2.5 mg/kg), and Rac1 protein levels were determined in HP tissue samples by a western blot analysis (fi). The protein samples were equally loaded, confirmed with Ponceau S staining, and normalized to β-actin. Rac1 mRNA levels (fii) were determined in samples exposed to saline and ethanol (8 h) with and without RV by qPCR analysis. Hprt mRNA was used as the internal control for normalization of Rac1 mRNA. Mice were exposed to a high dose of ethanol for 8 h after preadministration (30 min) with vehicle or SR (1 mg/kg), and Rac1 protein levels were determined in HP tissue samples by a western blot analysis (g). The protein samples were equally loaded, confirmed with Ponceau S staining, and normalized to β-actin (*p < 0.05 vs. S; #p < 0.05 vs. E). Error bars, SEM (n = 12 pups/group)