Fig. 5
Inhibition of Hdac3 improves PPARα responses in livers of Vps15-LKO mice. a The OCR measured by SeaHorse Bioanalyzer in primary hepatocytes 48 h post-transduction with adenoviral vectors expressing GFP, shRNAVps15 or shRNAVps15 + shRNAHdac3 under basal conditions and in response to sequential treatment with indicated reagents. Dashed lines indicate the time of the addition of each reagent. Representative experiment of three independent hepatocyte cultures is presented. Quantification of basal respiration, ATP production and maximal respiratory capacity are shown on the graphs (middle panel). Relative transcript levels of Vps15 and Hdac3 is shown on right panel. Data are means ± SEM (n = 3, P < 0.05 *: vs GFP-infected cells, #: vs shRNAVps15-infected cells, two-tailed, unpaired Student’s t test). b Immunoblot analysis of chromatin extracts from the liver tissue of six week old random-fed Vps15f/f and AlbCre+;Vps15f/f mice that were treated for two weeks with VPA incorporated in food or control chow food. Densitometric analyses of H3K27Ac levels normalised to total H3 levels presented as folds over Vps15f/f-chow condition. Data are means ± SEM (n = 4 for Vps15f/f and AlbCre+;Vps15f/f chow, n = 6 AlbCre+;Vps15f/f VPA, P < 0.05 *: vs Vps15f/f, #: vs chow, two-tailed, unpaired Student’s t test). c Relative mRNA expression levels of Aox in the livers of mice treated as in b. Data are means ± SEM (n = 6 for Vps15f/f and AlbCre+;Vps15f/f chow, n = 5 Vps15f/f VPA, n = 6 AlbCre+;Vps15f/f VPA, P < 0.05 *: vs Vps15f/f, #: vs chow, two-tailed, unpaired Student’s t test). d Immunoblot analysis of total protein liver extracts of mice treated as in b using indicated antibodies. Densitometric analyses of protein levels normalised to Pras40 levels presented as folds over Vps15f/f-chow condition. Data are means ± SEM (n = 4 for Vps15f/f and AlbCre+;Vps15f/f chow, n = 6 AlbCre+;Vps15f/f VPA, P < 0.05 *: vs Vps15f/f, #: vs chow, two-tailed, unpaired Student’s t test). e Heat map showing relative levels of Acetyl-CoA and Acyl-carnitine metabolites measured by mass spectrometry in the livers of mice treated as in b. Each column corresponds to an individual animal and the colour of the cell indicates the relative content of the metabolite (from blue to red). Data are means ± SEM (n = 4 for Vps15f/f and AlbCre+;Vps15f/f chow, n = 5 Vps15f/f VPA, n = 6 AlbCre+;Vps15f/f VPA, P < 0.05 *: AlbCre+;Vps15f/f vs Vps15f/f, #: AlbCre+;Vps15f/f VPA vs AlbCre+;Vps15f/f chow, two-tailed, unpaired Student’s t test)
