Fig. 6: TMEM165 is present on the lysosomal limiting membrane.

a Representative (n = 3) confocal images of live HeLa-TMEM165-mCherry cells transfected with a Golgi marker EGFP-RAB6 (top), or stably expressing Lyso-GFP (middle and bottom). Yellow arrows point to vacuolin-enlarged lysosomes with both Lyso-GFP and TMEM165-mCherry on limiting membranes. DNA is visualized by Hoechst staining. White squares mark the area shown in enlarged images. r, Pearson’s colocalization coefficient (n = 20). Scale bar, 10 μm. b Representative (n = 3) confocal images of live HeLa-TMEM165-mCherry-Lyso-EGFP cells with indicated treatments or genetic alteration. r, Pearson’s colocalization coefficient (n = 20). Scale bars, 10 μm. c Representative (n = 3) immunoblots of indicated proteins from purified lysosomes (Lys), light membrane fractions (LMF) and whole cell lysates (WC) of HeLa cells. When indicated, 10 nM concanamycin A (ConA) was added to all buffers used for the extraction. d Representative (n = 3) immunoblots of indicated proteins in WT and TMEM165-KO MDA-MB-468 clones treated with 1 μM MnCl₂ for indicated times (left), and relative quantification of fully glycosylated LAMP2 (right). e Cytosolic Ca²⁺ concentration analyzed by Fluo-4-AM FLI in WT and TMEM165- KO MDA-MB-468 clones treated with DMSO or 15 μM ebastine for the last 1 h of the 48 h treatment with 1 μM MnCl₂. Bars, SD of three independent experiments with at least 5000 cells analyzed in each sample. f Cytosolic pH analyzed by pHrodo-AM FLI in WT and TMEM165- KO MDA-MB-468 clones treated with DMSO, 6 μM terfenadine, or 15 μM ebastine for the last 1 h of the 48 h treatment with 1 μM MnCl₂. Bars, SD of three independent experiments with 30 randomly chosen cells analyzed in each sample. ***P < 0.001 as analyzed by one-way Anova with Tukey’s multiple comparison (d right) or two-way Anova with Dunnett’s multiple comparison (e, f).