Fig. 1: TMEM175 acidifies the perilysosomal area.
a Schematic presentation of Lyso-mKeima (top) and representative confocal images of live HeLa-Lyso-mKeima cells stained with Lysotracker® Green (bottom). r, Pearson’s colocalization coefficient (n = 20). Scale bars, 10 μm. b The standard curve for fluorescence intensity (FLI) ratios (Ex 585 nm/405 nm; Em 620 nm) of mKeima at pH ranging from 6.0 to 7.5. See Supplementary Fig. 1d for corresponding images. c Cytosolic and perilysosomal pH values of indicated cell lines. d Representative (n = 3) immunoblots of indicated proteins in HeLa cells treated with control (CTL) or TMEM175 siRNAs for 72 h. e Perilysosomal pH analyzed by Lyso-mKeima (left) and lysosomal pH analyzed by LysoSensorTM Yellow/Blue FLI (right) in HeLa cells treated with indicated siRNAs for 72 h. f Relative volumes of acidic compartments in HeLa cells treated with Con A for 1 h, stained with Lysotracker green, and analyzed by flow cytometry (left), and mKeima FLI in HeLa-Lyso-mKeima cells treated for 72 h with indicated siRNAs and with either DMSO or 10 nM Con A for 1 h (right). See Supplementary Fig. 1h for flow cytometry gating. g Representative images of live HeLa-Lyso-mKeima cells treated with DMSO or 100 μM Arachidonic acid for 15 min (left) and corresponding mKeima FLIs (right). h Relative mKeima FLIs in HeLa-Lyso-mKeima cells treated for 72 h with indicated siRNAs and with either DMSO or 100 μM Arachidonic acid for the last 20 min. Bars, SD of three independent experiments with 10 (g) 30 (b, c, e-left, f-right, h) or ≥10000 (e-right, f-left) randomly chosen cells analyzed in each sample. *P < 0.05; **P < 0.01; ***P < 0.001 as analyzed by one-way Anova with Tukey’s multiple comparisons (e, f left), two-way Anova with Dunnett’s multiple comparison (c, f right, h), or two-tailed, homoscedastic t-test (g).
