Fig. 1
Mitochondrial gene transcription programme is defective in Vps15-LKO mice. a Functional annotation clustering by Database for Annotation, Visualization and Integrated Discovery (DAVID) bioinformatic tool of significantly downregulated genes in the microarray analyses of liver tissue after acute Vps15 depletion. Percentage of genes among downregulated genes attributed to listed processes is indicated. Liver tissue was harvested ten days after transduction with adenoviral vectors expressing Cre recombinase to deplete Vps15 or GFP control protein. Significantly modified genes are listed in Supplementary Data 1. Functional annotation clustering results are listed in Supplementary Data 2. b Relative transcript levels of genes implicated in mitochondrial DNA replication, transcription, respiratory chain subunits and metabolic enzymes in livers of control and Vps15-LKO mice analysed by RT-qPCR. Data are means ± SEM (n = 6–8 for Vps15f/f, n = 5 for AlbCre+;Vps15f/f, P < 0.05 *: vs Vps15f/f mice, two-tailed, unpaired Student’s t test). c Immunofluorescent analyses of Tom 40 in control and Vps15-null primary hepatocytes. Cells were PFA fixed and stained with anti-Tom 40 antibody, secondary anti-rabbit IgG Alexa Fluor 568 antibody was used for detection. Scale bar: 50 µm. d The total respiratory chain activity normalized to total protein content measured in liver tissue extracts of Vps15f/f and AlbCre+;Vps15f/f mice. Data are means ± SEM (n = 4 for Vps15f/f and AlbCre+;Vps15f/f, P < 0.05 *: vs Vps15f/f, two-tailed, unpaired Student’s t test). e The oxygen consumption rate measured by SeaHorse Bioanalyzer in primary Vps15f/f hepatocytes 48-h post-transduction with adenoviral vectors expressing GFP, Cre or shRNAVps15 under basal conditions (initial rates) and in response to sequential treatment with Oligomycin (respiration associated with ATP production), FCCP (maximal respiration), and Rotenone/Antimycin A (non-mitochondrial respiration). Dashed lines indicate the time of the addition of each reagent. Representative experiment of five independent hepatocyte cultures is presented. Quantification of basal respiration, ATP production and maximal respiratory capacity are shown on the graphs (right panel). Data are means ± SEM, P < 0.05 *: vs GFP-infected primary hepatocytes, two-tailed, unpaired Student’s t test. Lower panel shows the control immunoblot analysis of total protein extracts of hepatocytes using indicated antibodies. The immunoblot with anti-actin antibody served as a loading control
