Tem increases autophagic flux through TRPML1.

(A) Tem (5 μM, 3 h) induced TFEB (green) nuclear translocation in TRPML1 stable cell lines (TRPML1 HEK Tet-On) upon Dox (1 μg/ml, overnight) induction. GPN (200 μM, 2 h) was used as a positive control due to its consistent activation on TFEB in HEK cells [37]. Nuclei were labelled with DAPI (red, pseudo-color). Scale bar = 10 μm. (B) Tem (10 μM, 9 h) dramatically increased LC3-II levels in Dox-induced cells, which was blocked by ML-SI3 (10 μM). (C) Summary of Tem effects on LC3-II levels (normalized with GAPDH expression). (D) Dose-dependent effects of Tem (0.5, 1, 2.5, 5, and 10 μM; 9 h treatment) on LC3-II expression levels in Dox-induced TRPML1 stable cells. (E) Quantification of dose-dependent Tem effects shown in D. (F) Tem (10 μM, 9 h) elevated LC3-II levels in WT but not ML1−/− human fibroblasts. Baf-A1 treatment increased LC3-II levels in both WT and ML1−/− cells. Tem effects in WT cells were blocked by ML-SI3. (G) Quantification of Tem effects on LC3-II levels in fibroblasts. Data shown in C, E, and G were obtained from at least 3 independent experiments and are presented as mean ± SEM. The individual data of C, E, and G can be found in S1 Data. *P P P −/−, Mucolipidosis IV; ML-SI3, TRPML1 synthetic inhibitor 3; Tem, temsirolimus; Tet-On, Tetracycline-On; TFEB, transcription factor EB; TRPML1, transient receptor potential channel mucolipin 1; WT, wild type.