Extended Data Fig. 3: Aldometanib activates the lysosomal pool of AMPK.
From: The aldolase inhibitor aldometanib mimics glucose starvation to activate lysosomal AMPK
a, b, e-h, aldometanib activates AMPK via the lysosomal pathway. HEK293T cells (a), mouse primary myocytes (b), AXIN-/- MEFs (e), HEK293T cells with ATP6v0c knocked down (f), PEN2-/- MEFs (g), or ATP6AP1-/- MEFs with the Δ420-440 mutant of ATP6AP1 re-introduced (h) were treated with aldometanib at indicated concentrations for 2 h, and the levels of p-AMPKα and p-ACC were determined by immunoblotting. c, aldometanib inhibits mTORC1. MEFs, HEK293T, primary hepatocytes and primary myocytes were treated with 5 nM aldometanib for 2 h, and the activity of mTORC1, as assessed by the phosphorylation levels of its substrate S6K (p-S6K), was determined through immunoblotting. d, i, aldometanib triggers the lysosomal pathway. MEFs were treated as in c, followed by immunoprecipitation (IP) of AXIN, or purification of lysosome (as in Fig. 1p). j-l, aldometanib does not inhibit, and even slightly enhances glycolysis. Wildtype MEFs (j, k), AMPKα-/- MEFs (l), and HEK293T cells (k) were treated with aldometanib for 2 h (j) or 4 h (k, l), followed by determining glycolytic rates, either through labelled metabolites tracing [j, using the levels of labelled (M + 6) FBP as an indicator of glycolytic rate] or lactate production (k). Results are shown as mean ± s.d.; n = 6 (j) or 4 (k, l) dishes of cells for each condition; and P value by one-way ANOVA followed by Dunn (j) or Tukey (k), or by two-way ANOVA, followed by Tukey (l). m, aldometanib activates AMPK without elevation of AMP/ADP. HEK293T cells (left panel) or primary myocytes (right panel) were treated with aldometanib at indicated concentrations for 2 h, followed by determining the AMP:ATP and ADP:ATP ratios. Results are shown as mean ± s.e.m.; n = 4 dishes of cells for each treatment, and P value by one-way ANOVA followed by Dunnet (HEK293T) or Dunn (myocytes). n, The gating strategy for quantifying the populations of FITC-labelled cells. Experiments in this figure were performed three times, except k and l four times.
