|
Deletion of TBC1D23 specifically impairs Golgi-AMPK activation. a HepG2 cells were transiently transfected with Flag-LKB1 for 24 h, and incubated with or without glucose-free medium for 2 h. Cells were stained with antibodies against Flag and golgin-97, a Golgi marker. Scale bar, 10 μm. Mander’s coefficient 2 (golgin-97 overlapping with Flag-LKB1) were graphed as mean ± SD. p value: 2.41e−030. b HepG2 cells transiently transfected with Flag-AMPKα1 were subjected to glucose starvation for 2 h. Scale bar, 10 μm. Mander’s coefficient 2 (golgin-97 overlapping with Flag-AMPKα1) were graphed as mean ± SD. Each dot represents Manders’ coefficients from one cell (a, b). c Schematic diagram of subcellular compartment-specific biosensor osABKAR. d, e WT and TBC1D23 KO HepG2 cells transiently transfected with Golgi-ABKAR (d) or Mito-ABKAR (e) were incubated with glucose-free medium for 2 h and 4 h, respectively. The FRET/CFP ratio of WT cells incubated with DMEM was set as 1, and FRET/CFP ratio of other groups was normalized to WT cells incubated with DMEM. d WT Con vs KO Con: p value: 1.47e−011; WT GS vs KO GS: p value: 0.00e + 000; WT Con vs WT GS: p value: 0.00e + 000; KO Con vs KO GS, p value: 4.07e−005. e WT Con vs WT GS: p value: 1.91e−005. f WT and TBC1D23 KO HepG2 cells were treated with or without 1 mM metformin for 2 h. Cells lysates were analyzed by immunoblotting. The graph shows the levels of pAMPK quantified by densitometry using Image J software and normalized to GAPDH. g, h WT and TBC1D23 KO HepG2 cells transiently transfected with Lyso-ExRai-AMPKAR for 24 h were incubated with or without glucose-free medium for 2 h. Representative images (g) and statistical analysis (h) of Lyso-AMPK activity measured by Lyso-ExRai-AMPKAR. Scale bar, 10 μm. Similar results were obtained in three independent experiments (a–h). a–h Statistical data are presented as mean ± SD. P values were determined by unpaired two-tailed Mann–Whitney test (a, b), or by two-way ANOVA, followed by Sidak’s test (d–f), or by Scheirer-Ray-Hare Test (h). Source data are provided as a Source data file.
|
