Extended Data Fig. 4: Golgi PtdIns4P regulates ATGL protein stability in the Golgi apparatus.
From: Glucose controls lipolysis through Golgi PtdIns4P-mediated regulation of ATGL
(a) Representative images of P4M-EGFP signal in the HEK293-AAV cells 12 h after glucose deprivation (n = 107 cells per group). Scale bar, 5 μm. (b) Representative images of SACM1L-EGFP signal in the HEK293-AAV cells after glucose deprivation. Scale bar, 5 μm. Three times repeated independently with similar results. (c) Determination of total PtdIns4P levels in the HEK293T cells 24 h after glucose deprivation (n = 9 independent experiments per group). (d) Immunoblot of ATGL and pAMPK in the HEK293T cells after treatment with AMPK inhibitor Compound C (Cpd C) and activator AICAR (n = 4 independent experiments per group). (e) PtdIns4P quantification via immunostaining in the HEK293-AAV cells after treatment with Cpd C and AICAR (n = 67 cells per group). Scale bar, 5 μm. (f–g) Quantification of PtdIns4P levels and Golgi area via immunostaining in the HEK293-AAV cells 72 h after PI4KB and SACM1L knockdown (n = 103 cells per group in f; n = 130 cells per group in g). Scale bar, 5 μm. (h) Quantification of ATGL transcript in the HEK293T cells 72 h after PI4KB and SACM1L knockdown (n = 4 independent experiments per group). (i–j) Quantification of PtdIns4P levels and Golgi area via immunostaining in the HEK293-AAV cells after 24 h treatment with 5 μM PIK93, 10 μM BF738735 and 10 μM UCB9608 (n = 136 cells per group in i; n = 100 cells per group in j). Scale bar, 5 μm. (k) Immunoblot of ectopically expressed ATGL in the HEK293T cells 24 h after treatment with 5 μM PIK93, 10 μM BF738735 and 10 μM UCB9608 (n = 3 independent experiments per group). Results are shown as mean ± s.e.m. and analyzed using two-tailed unpaired t-test (c and d (Ctr and AICAR)), two-tailed Mann-Whitney test (a, d (DMSO and Cpd C) and e), one-way ANOVA method with Dunnett correction for multiple comparisons between control and other groups (h and k) and Kruskal-Wallis test with Dunn’s correction for multiple comparisons between control and other groups (f, g, i and j). Source numerical data and unprocessed blots are available in source data.
