Fig. 1: Aldometanib activates lysosomal AMPK.
From: The aldolase inhibitor aldometanib mimics glucose starvation to activate lysosomal AMPK
a,b, Aldometanib inhibits the enzymatic activity of purified aldolase. Enzymatic activities of rabbit aldolase are shown in b as the mean ± s.e.m.; n = 4 biological replicates for each concentration of aldometanib (see structure in a), and P values were determined by one-way analysis of variance (ANOVA), followed by Tukey’s test. c, Aldometanib is able to bind aldolase. Representative sensorgrams of SPR assays in which aldometanib were incubated with rabbit aldolase immobilized on a CM5 chip (solid line), and fitted curves for each measurement (dashed line) are shown. RU, response unit. d, Aldometanib prevents FBP from binding to aldolase. Rabbit aldolase was incubated with aldometanib, followed by determining the potential FBP and dihydroxyacetone phosphate (DHAP) binding by MS as described previously64. e,h, The Arg43 residue of aldolase is required for aldometanib binding. Enzymatic activities of bacterially expressed ALDOA-p.Arg43Ala (e), and AMPK activation in ALDO-TKD MEFs reintroduced with ALDOA-p.Arg43Ala (h) were determined. Data are the mean ± s.d. (e) or s.e.m. (h; quantification of immunoblots); n = 3 biological replicates for each condition, and P values were by one-way (e) or two-way (h) ANOVA, followed by Tukey’s test (e and h). f,g,l–o, Aldometanib activates AMPK via the lysosomal pathway. Wild-type MEFs (f) and MEFs LKB1−/− (l), CaMKK2−/− (m), TRPV1-4-QKO (n), LAMTOR1−/− (o) or mouse primary hepatocytes (g) were treated with aldometanib. Quantification data for immunoblots are the mean ± s.e.m., n = 4 (p-ACC/ACC of g) or 3 (others) dishes of cells for each condition, with P values calculated by two-way ANOVA followed by Tukey’s test (n and o), or by one-way ANOVA, followed by Tukey’s test (f and g; comparison between control and aldometanib-treated groups). i–k, Aldometanib inhibits TRPVs and induces AXIN lysosomal translocation. MEFs were infected with TRPV4-GCaMP6s (i), pre-labelled with (j) or without (k) LysoSensor, and then treated with aldometanib, followed by determining the fluorescence intensity of TRPV4-GCaMP6s (i), LysoSensor (j) or staining AXIN and the lysosomal marker LAMP2 (k). Results are the mean ± s.d. (i) or s.e.m. (others); n = 19, 35, 18, 20, 19 and 18 cells from 5, 2, 4, 3, 4 and 3 dishes/fields for i, j, control of wild-type MEFs of k, aldometanib treatment of wild-type MEFs of k, control of LAMTOR1−/− MEFs and aldometanib treatment of LAMTOR1−/− MEFs, respectively; and P values were determined by two-sided Student’s t-test with Welch’s correction (i and j), or by two-way ANOVA followed by Tukey’s test (k). p, Aldometanib specifically activates lysosomally localized AMPK. MEFs or primary hepatocytes were treated with aldometanib, or AICAR (representing severe stress9), followed by analysis of p-AMPKα in lysosomes (Lyso), cytosol (Cyto), mitochondria (Mito) and total cell lysates (TCL). Quantification data are the mean ± s.e.m., from n = 3 biological replicates, with P values calculated by one-way ANOVA, followed by Tukey’s test (compared as in f). Experiments were performed three times, except in b and e, which were performed four times. WT, wild type.
