Fig. 2: PARKIN down regulation and impairment of mitophagy in DS fibroblasts.

a, b Whole-cell extracts from 2 N and DS cells were analyzed by western blot for PARKIN and PINK1. Each quantitative data was normalized with ACTIN. (n ≥ 3). c 2 N and DS cells were assessed for PARKIN and PINK1 mRNA by quantitative real-time PCR. mRNA levels were normalized to ACTIN mRNA, used as internal control. Data display the fold-changes of PARKIN, PINK1 and SQSTM1 (p62) mRNAs relative to control cells (n ≥ 3). d Mitochondrion/cytosol fractionation was performed to assay the presence of PARKIN and LC3-II within the mitochondrial fraction. VDAC1 and COXIV were used as markers of purity for mitochondrial fraction. βIII-TUBULIN was used as a marker of the cytosolic fraction. e Quantitative data of LC3-II in the mitochondrial fraction, normalized with VDAC1 (n = 5). f Mitochondrial fractionation was performed to assess the accumulation of p62, LC3-II, NDP52 and OPTINEURIN (OPTN) within the mitochondria in untreated and treated fibroblasts with a lysosomal-inhibiting agent, Bafilomycin A (Bafil., 10 nM) for 6 h. Each protein was normalized with VDAC1 with quantitation in (g). (n ≥ 3) h 2 N and DS cells were treated with Concanamycin A (ConA, 50 nM) for 2 h to block lysosome function. The cells were fixed and immunostained for LC3 and TOM20 in order to evaluate mitophagy. White arrows point to the co-localization of puncta into the high magnification image of the boxed area. Scale bar 10 μm. The graph shows the percentage of LC3/TOM20 colocalization calculated by JACoP plugin of ImageJ. Quantitation based on a minimum of 50 cells per conditions from three independent experiments. i GSEA analysis of transcriptomic changes comparing DS fibroblasts to 2 N fibroblasts, using a list of transcriptional factors that have been described to be involved in mitophagy^46,47 (for list of genes, see Supp. Table 1). As described in Fig. 1d, the vertical bars represent genes ranked horizontally by moderated t-statistics; Pink, blue and grey shaded rectangles upregulated, downregulated and genes with no change respectively. The plot shows significant downregulation of TFs involved in mitophagy in DS compared to 2 N fibroblasts (p < 0.001; FDR < 0.001). j Volcano plots for whole transcriptomes of differentially expressed genes between DS and 2 N fibroblasts, with TFs involved in mitophagy, used in (I) for GSEA analysis highlighted with labels. Statistical analysis was performed using Student’s T-test or one-way ANOVA with Tukey’s multiple comparisons test. (**p < 0.01; ***p < 0.001). Immunoblots reported are from one representative experiment