Fig. 3: Acute Sac1 depletion results in TGN deacidification caused by V-ATPase disassembly.
From: Control of Golgi- V-ATPase through Sac1-dependent co-regulation of PI(4)P and cholesterol
a Violin plot showing the distribution of TGN pH values of Sac1-degron A431 cells transiently transfected with GalT-RpHLuorin2 and treated with DMSO or IAA for 0 to 3 h. n = 3 independent experiments. One-way ANOVA with Tukey’s multiple comparisons test. n.s. p > 0.9999, p = 0.9848, * p = 0.0480, **** p < 0.0001. b Violin plot depicting the distribution of TGN pH values of Sac1-degron A431 cells treated for 3 h with DMSO or IAA with or without 50 µM EN6. n = 3 independent experiments. One-way ANOVA with Tukey’s multiple comparison test. n.s. p = 0.4775, ** p = 0.0039, **** p < 0.0001. c Western blot analysis of Sac1-degron A431 cells treated for 4 h with DMSO or IAA with or without 50 µM EN6. Immunoblotted for TGN46, B4GALT1 and miniIAA7 (Sac1). d Bar graph showing average normalized immunoblot intensities from (c). n = 4 independent experiments. One-way ANOVA with Tukey’s multiple comparisons test. TGN46: *** p = 0.0004, p = 0.0002, **** p < 0.0001. B4GALT1: * p = 0.0127, ** p = 0.0022, p = 0.0026. miniIAA7: *** p = 0.0001, **** p < 0.0001. n.s. p = 0.7763. e Representative confocal images of Sac1-degron A431 cells treated for 4 h with DMSO or IAA with or without 50 µM EN6. Stained for DAPI, Golgin-97 and TGN46. f Violin plot depicts the distribution of the TGN46 intensity in the Golgi area calculated from (e). n = 3 independent experiments. One-way ANOVA with Tukey’s multiple comparisons test. n.s. p = 0.6182, **** p < 0.0001. g Cartoon illustrating the reversible assembly of the V0 and V1 regions of the V-ATPase complex. Upon assembly, ATP hydrolysis of the V1 region drives the proton pumping ability of the V0 region. h Representative fluorescence micrographs of Sac1-degron A431 cells treated with DMSO for 4 h or IAA for 2 or 4 h and stained for ATP6V0a2 or ATP6V1A. i Violin plot showing the distribution of the Pearson’s correlation coefficient between ATP6V0a2 and ATP6V1A from (h). Representative data from one of three different experiments. One-way ANOVA with Dunnett’s multiple comparisons test. **** p < 0.0001. Representative confocal images of Sac1-degron A549 cells treated with DMSO or IAA for 2 h and stained for ATP6V1A and Golgin-97 (j) or ATP6V1A and LAMP2 (l). k, m Violin plot showing the Pearson’s correlation coefficient between ATP6V1A and Golgin-97 (j) and ATP6V1A and LAMP2 (l). n = 3 independent experiments. Two-tailed unpaired Student’s t-test. **** p < 0.0001, n.s. p = 0.1287.
