Fig. 7: Lysosomal exocytosis, pH and TRPML1 activity in WT and Trpml3^−/− AMΦ.

a Lysosomal exocytosis experiments measuring hexosaminidase release from WT and Trpml3^−/− AMΦ. Maximum effects were obtained with ionomycin (4 µM). TRPML3 activator ML3-SA1 elicited no significant effects in both WT and Trpml3^−/− AMΦ. Each dot corresponds to one biologically independent experiment. Average values are mean values ± SEM, each. b Cartoon illustrating LAMP1 translocation assay shown in c, d. Upon lysosomal exocytosis the lysosomal protein LAMP1 is detected on the plasma membrane (PM) by anti-LAMP1 followed by Alexa Fluor 488-conjugated secondary antibody. c Representative images of LAMP1 translocation assay using WT and Trpml3^−/− AMΦ. Shown are results obtained after 120 min treatment with DMSO, ML3-SA1 (30 µM), or 10 min treatment with ionomycin (4 µM). Scale bar 10 µm. d Quantification of experiments as shown in c (mean ± SEM from 3 biologically independent experiments, each). e Fura-2 calcium imaging experiments using HEK293 cells expressing human or murine TRPML1(NC), TRPML2 or TRPML3, respectively, indicating the specific levels of activation. Channels were stimulated with either ML1-SA1 ( = EVP-169) or ML-SA1 (10 µM, each). Shown are average values (mean ± SEM). Each dot represents one biologically independent experiment with 10–20 cells, each. ****p < 0.0001; Two-way ANOVA followed by Tukey’s multiple comparisons test. f Chemical structures of ML-SA1 and its derivative ML1-SA1 ( = EVP-169). g, h Quantification (g) and representative currents (h) from YM201636-enlarged LE/LY isolated from WT or Trpml3^−/− AMΦ, elicited by an application of 10 µM ML1-SA1. Each dot corresponds to one biologically independent experiment. Average values are mean values ± SEM, each. *p < 0.05, ***p < 0.001; One-way ANOVA followed by Tukey’s post hoc test. i Results obtained from endolysosomal pH measurements using WT or Trpml3^−/− AMΦ. Measurements were performed by ratiometric fluorescence imaging with Oregon Green^22,71. Data are mean ± SD. j Mean endolysosomal pH values (mean ± SD) in WT and Trpml3^−/− AMΦ were calculated using the calibration curves presented in i (n = 4, each). Source data are provided as a Source Data file.