Fig. 4: Alterations in glycolysis in ATG5 cKO PCs correlate with their elevated GLUT2 levels.
From: Autophagy regulator ATG5 preserves cerebellar function by safeguarding its glycolytic activity
a,b, Representative confocal images (a) of 2-NBDG uptake in WT (108 cells) and ATG5 cKO (139 cells) PCs and its fluorescence-based analysis (b). Scale bars, 50 µm. N = 4 mice. P < 0.0001. c–e, FRET (c), ratiometric analysis (d) and area under the curve (AUC) (e) of Laconic in PCs from WT and ATG5 cKO OTCs, perfused with 5 µM pyruvate for 5 min. Baseline: WT N = 3 (51 cells), cKO N = 4 (84 cells); pyruvate: WT N = 3 (59 cells), cKO N = 4 (70 cells). Scale bars, 20 µm. WTbaseline versus cKO baseline: P < 0.0001; WTbaseline versus WTpyruvate: P < 0.0001, cKObaseline versus cKOpyruvate: P < 0.0001. f,g, FRET (f) and ratiometric (g) analysis of ATeam1.03YEMK in PCs from WT and ATG5 cKO OTCs, perfused with 1.5 µM oligomycin A for 30 min. WT N = 5 (46 cells), cKO N = 5 (35 cells). Scale bars, 50 µm. h, Ratiometric analysis of ATeam1.03YEMK in WT and ATG5 cKO PCs (30 cells, N = 5 per genotype) after perfusion with 1.5 µM oligomycin A and 10 mM 2-DG for 30 min. i, AUC of ATeam1.03YEMK in WT and ATG5 cKO PCs. mTFP, monomeric teal fluorescent protein; mVenus, monomeric Venus protein; eCFP, enhanced cyan fluorescent protein. Baseline: WT 76 cells N = 10, cKO 70 cells N = 10; oligomycin A: WT 46 cells N = 5, cKO 35 cells N = 5; oligomycin A + 2-DG: WT 30 cells N = 5, cKO 35 cells N = 5. WTBaseline versus WTOligomycinA+2-DG: P < 0.0001; WTBaseline versus cKOBaseline: P < 0.0029; cKOBaseline versus cKOOligomycinA+2-DG, P < 0.0001; WTBaseline versus WTOligomycinA: P = 0.004; WTOligomycinA versus cKOOligomycinA: P < 0.0001; cKOBaseline versus cKOOligomycinA+2-DG, P < 0.0001. j–m, GLUT2 levels in WT and ATG5 cKO PCs at 1 M (j) and 3 M (k). Scale bars, 50 µm; magnified image scale bars, 10 µm. N = 4 for 1 M per genotype and 3 M WTs, N = 6 for 3 M ATG5 cKO soma and N = 5 for dendrites. l, Quantitative analysis of GLUT2 levels in PC soma; WT1M versus cKO1M: P = 0.031, WT3M versus cKO3M, P = 0.022. m, Quantitative analysis of GLUT2 levels in PC dendrites; WT3M versus cKO3M: P = 0.022. n,o, Western blot (n) and analysis (o) of GLUT2 surface biotinylation in 3 M WT and ATG5 cKO cerebellum. N = 4 mice per genotype, P = 0.032. Western blot of E-cadherin shows pooled WT and ATG5 cKO samples and is not used for quantification in o or directly comparable to the GLUT2 and β-actin western blot shown above. p, GLUT2 surface levels in WT and ATG5 cKO PCs. White circles outline PCs. Scale bars, 50 µm; magnified image scale bars, 10 µm. q, Quantitative analysis of images in p. N = 3 mice per genotype, P = 0.027. m.g.v., mean grey value. Squares in j, k and p indicate regions magnified. A coloured bar in c and f indicates the relative FRET signal. Statistical significance in e, i and m was determined by two-way ANOVA followed by Holm–Sidak multiple-comparisons test. Statistical significance in b, o and q was determined by two-tailed unpaired t-test. All graphs show the mean ± s.e.m. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.
