Extended Data Fig. 5: Effects of Cytosolic AcCoA reduction on mitochondrial functions and PINK1-Parkin pathway.
From: Cytosolic acetyl-coenzyme A is a signalling metabolite to control mitophagy
HeLa cells were cultured with SM, HC (20 mM), SB (100 μM), BTC (5 mM), or CCCP (10 μM) for 16 h in (a-h), CCCP (10 μM) for 1 h in (i, j), MitoBloCK-6 (MB-6) (100 μM) for 6 h in (d), or actinomycin D (10 μM) for 16 h in (e). a, SM and SLC25A1 or ACLY inhibition do not decrease Mito membrane potential according to flow cytometry analysis of TMRM staining. Left panel: histogram plot of the TMRM fluorescence. Right panel: relative quantitative analysis of the fluorescence level of TMRM. Data shown as the mean ± s.e.m. Ordinary one-way ANOVA and Dunnett’s multiple comparisons test. n = 3 biological replicates. b, Effects of SM and SLC25A1 or ACLY inhibition on Mito ROS production according to flow cytometry analysis of Mito ROS production by MitoSOX staining. Left panel: histogram plot of the MitoSOX fluorescence. Right panel: relative quantitative analysis of the fluorescence level of MitoSOX. Data shown as the mean ± s.e.m. Ordinary one-way ANOVA and Dunnett’s multiple comparisons test. n = 3 biological replicates. c, Effects of SM and SLC25A1 or ACLY inhibition on intracellular ATP level according to luminescence determination. Data shown as the mean ± s.e.m. Kruskal-Wallis test and Dunn’s multiple comparisons test. n = 4 biological replicates. d, Effects of SM and SLC25A1 or ACLY inhibition on Mito protein import. HeLa-rtTA cells transfected with the vector expressing doxycycline (dox)-inducible MTS-EGFP and treated with dox (0.25 μg per ml) for 6 h before stimulation. Total Mito were stained using Mitotracker (purple). Left panel: representative cell images of MTS-EGFP (green) localization compared with mitochondria (purple) shown. Scale bar, 10 μm. Right panel: quantitative analysis of the proportion of cells with improperly localized MTS-EGFP (localized in the cytosol or nucleus). n = 26 (Mock), n = 32 (SM), n = 27 (HC), n = 28 (SB), n = 27 (BTC) and n = 29 (MB-6) images. Data shown as the mean ± s.e.m. Kruskal-Wallis test and Dunn’s multiple comparisons test. e, SM and SLC25A1 or ACLY inhibition do not induce mitochondria permeability transition determined by Cytochrome c release. Cyto fractions were isolated after the indicated treatment, and Cytochrome c level was determined by immunoblotting. n = 3 biological replicates. f,g, Effects of SM and SLC25A1 or ACLY inhibition on oxygen consumption rate (OCR) at routine rates (f), cell death according to analysis of LDH release (g). n = 3 biological replicates. Data shown as the mean ± s.e.m. Ordinary one-way ANOVA and Dunnett’s multiple comparisons test. h-j, SM and SLC25A1 or ACLY inhibition do not induce PINK1 protein stabilization (h), Parkin’s E3 activity (i) and localization on mitochondria (j). n = 3 biological replicates (h). i,j, HeLa cells stably expressing GFP-Parkin were generated, and after treatments, immunoblotting analyses of ubiquitinated GFP-Parkin were determined (i) and Mito marker TOM20 was stained (j). n = 3 biological replicates (i). j, Left panel: representative cell images of colocalization of GFP-Parkin with TOM20 are shown Scale bar, 10 μm. Right panel: the statistical analysis of Parkin colocalization with mitochondria. n = 10 images for all groups except for the Mock and CCCP groups (n = 11 images). Data shown as the mean ± s.e.m. Kruskal-Wallis test and Dunn’s multiple comparisons test. k, Volcano plot of the whole genome depleted genes in mitophagic cells treated with HC. Autophagy-related genes were highlighted in red. l, Gene ontology analysis of Fig. 1e. The P values were calculated using MAGeCK software (k) or Metascape database (l).
