Fig. 6. TRPML1 activation with ML-SA1 promotes lysosomal TRPML1-mediated Ca²⁺ release and nuclear translocation of TFEB and consequent lysosomal biogenesis in U-loaded renal epithelial HK-2 cells.

For experiments a, b, HK-2 cells were treated with U at 0, 100, and 600 μM for 24 h. After washout of U, cytosolic Ca²⁺ was measured after treatment with ionomycin (2 μM) followed ML-SA1 (10 μM) by Fluo-4 imaging. For experiments c–l, HK-2 cells or HK-2 cells transfected with either TRPML1 shRNA or PPP3CB siRNA or corresponding empty vector plasmid or scramble control siRNA were exposed to U at 0, 100 and/or 600 μM for 24 h. After washout of U, the cells were treated with vehicle or ML-SA1 (10 μM) for 30 min and then analyzed. a ML-SA1–induced lysosomal Ca²⁺ release in control and U-loaded HK-2 cells treated with 100 and 600 μM U for 24 h. b Quantification of cytosolic Ca²⁺ peak values shown in a. c, d Western blotting analysis of TFEB in HK-2 cells after U exposure and ML-SA1 treatment. e Representative images of immunofluorescence staining of TFEB (green) in HK-2 cells after U exposure and ML-SA1 treatment. Images share the same scale bar (20 μm). f Quantitative analysis of nuclear TFEB under various treatment conditions shown in e. g, h Western blotting analysis of LAMP-1 and TRPML1 in HK-2 cells after U exposure and ML-SA1 treatment. i, j Quantitative analysis of nuclear TFEB in TRPML1- or PPP3CB-knockdown HK-2 cells after U exposure and ML-SA1 treatment. Representative images of immunofluorescence staining of TFEB (green) are shown in Supplementary Fig. 8e, f. k, l Western blotting analysis of p-S6K and S6K in HK-2 cells after U exposure and ML-SA1 treatment. Iono: ionomycin. f, i, j More than 200 cells were analyzed in each sample. Data represent mean ± SD. n = 3 independent experiments. Statistical significance was evaluated by one-way ANOVA with LSD’s post hoc test (b, d, f, h–j, l). Source data are provided as a Source Data file.