Extended Data Fig. 1: Mild starvation induces mitophagy in an AMPK- and mTOR-independent manner.
From: Cytosolic acetyl-coenzyme A is a signalling metabolite to control mitophagy
a-c, The characterization of fasting mice. 6–8-week-old male mice were ad libitum-fed or fasted overnight for 16 h with free access to water. The mice body weight (a), n = 6 mice per group; the glucose level (b), n = 5 mice per group; the serum level of amino acids (c), the indicated amino acids measured by mass-spectrometry while the cysteine level was too low to be detected (valine, leucine and isoleucine were analysed by GC-MS while other amino acids analysed by LC-MS). n = 4 mice per group. Data shown as the mean ± s.e.m. Unpaired two-tailed Student’s t-test. d-f. SM induces mitophagy in a cell-line-dependent manner. Total cell or Mito lysates as indicated for immunoblotting (d), cells expressing mt-Keima reporter for flow cytometry analysis (e) or qPCR analysis of mtDNA/nDNA (f). n = 3 biological replicates. Data shown as the mean ± s.e.m. Unpaired two-tailed Student’s t-test for all groups except U-2 OS group in (e) (Mann-Whitney U-test) and MCF7 mtATP6 group in (f) (Welch’s t-test). g-i, Re-feed with NM could rescue SM-induced mitophagy. Total cell or Mito lysates from the indicated cells treated with SM for 16 h, followed by the NM for another 16 h for immunoblotting (g), cells expressing mt-Keima reporter for flow cytometry analysis (h) or qPCR analysis of mtDNA/nDNA (i). n = 3 biological replicates. Data shown as the mean ± s.e.m. Ordinary one-way ANOVA and Dunnett’s multiple comparisons test (h,i). j,k, RT-qPCR analysis of Mito biogenesis-related gene and nuclear-encoded Mito gene expression levels. Cells treated as indicated. n = 3 biological replicates. Data shown as the mean ± s.e.m. Unpaired two-tailed Student’s t-test (j) and Ordinary one-way ANOVA and Dunnett’s multiple comparisons test (k) except MCF7 HSP60 in (k) (Kruskal-Wallis test and Dunn’s multiple comparisons test). l,m, SM does not affect AMPK or mTOR pathway. The indicated cells were treated with SM, Torin 1 (100 nM) for 16 h or oligomycin (Oligo, 5 μM) for 5 min for immunoblotting. n = 3 biological replicates. n, Heat map of key intracellular metabolites level. Upper panel: the flow chart that the indicated cells were treated with complete medium (Mock) or SM for 16 h for Mass Spectrometry. Bottom panel: data calculated as relative metabolite ratios of SM/Mock and shown as mean values from 4 biological replicates for all metabolite except AcCoA (3 biological replicates). Data normalized to Actin. o, Metabolic enzyme protein expression level in different cell lines by immunoblotting. n = 3 biological replicates. p, SM reduces Cyto AcCoA level in a cell-line-dependent manner. Cells cultured with SM for 16 h for the relative abundance of Mito or Cyto AcCoA level by LC-MS. Data normalized to Tubulin (Cyto) or HSP60 (Mito) levels. n = 3 biological replicates. Data shown as the mean ± s.e.m. Unpaired two-tailed Student’s t-test.
