Extended Data Fig. 2: Cytosolic AcCoA level controls mitophagy in vitro.
From: Cytosolic acetyl-coenzyme A is a signalling metabolite to control mitophagy
a-d, SLC25A1 or ACLY inhibition decreases Cyto AcCoA level (a) and induces mitophagy (b-d). Indicated cells treated with HC (20 mM), SB (100 μM), BTC (5 mM) for 16 h for Mito or Cyto AcCoA analysis by LC-MS (a), immunoblotting (b), cells expressing mt-Keima reporter for flow cytometry analysis (c) or qPCR analysis of mtDNA/nDNA (d). n = 3 biological replicates. Data shown as the mean ± s.e.m. Ordinary one-way ANOVA and Dunnett’s multiple comparisons test (a,c,d). e-i, SLC25A1, ACLY or ACSS2 knockdown induces mitophagy. The indicated cells transfected with indicated siRNAs were cultured with or without Baf A1 (200 nM). Total cell or Mito lysates for immunoblotting (e,h), cells expressing mt-Keima reporter for flow cytometry analysis (f,i) or qPCR analysis of mtDNA/nDNA (g). n = 3 biological replicates. Data shown as the mean ± s.e.m. Ordinary one-way ANOVA and Dunnett’s multiple comparisons test (f,g) or unpaired two-tailed Student’s t-test (i). j,k, ACC1 or FASN knockdown does not affect mitophagy. The indicated cells were transfected with indicated siRNAs. Total cell or Mito lysates for immunoblotting (j), cells expressing mt-Keima reporter for flow cytometry analysis (k). n = 3 biological replicates. Data shown as the mean ± s.e.m. Ordinary one-way ANOVA and Dunnett’s multiple comparisons test (k). l-n, Ace blocks SM-induced mitophagy. The indicated cells cultured with SM for 16 h with or without 10 mM Ace. Total cell or Mito lysates for immunoblotting (l), cells expressing mt-Keima reporter for flow cytometry analysis (m) or qPCR analysis of mtDNA/nDNA (n). n = 3 biological replicates. Data shown as the mean ± s.e.m. Ordinary one-way ANOVA and Dunnett’s multiple comparisons test (m,n). o-q, Ace blocks ACLY knockdown-induced mitophagy. The indicated cells transfected with indicated siRNAs cultured with or without 10 mM Ace. Total cell or Mito lysates for immunoblotting (o), cells expressing mt-Keima reporter for flow cytometry analysis (p) or qPCR analysis of mtDNA/nDNA (q). n = 3 biological replicates. Data shown as the mean ± s.e.m. Ordinary one-way ANOVA and Dunnett’s multiple comparisons test (p,q).
