Rap and rapalogs induce TRPML1- and Ca²⁺-dependent TFEB nuclear translocation in TRPML1-overexpressing cells.

(A) Rap (5 μM) and Tem (5 μM) induced TFEB nuclear translocation in TFEB-GFP stable cells overexpressing mCherry-TRPML1 (asterisks). TFEB nuclear translocation was not seen with Zota (5 μM). Scale bar = 10 μm. (B) Summary of rapalog effects on TFEB nuclear translocation. (C) Blockade of Tem-induced TFEB translocation by ML-SI3 (10 μM). Scale bar = 10 μm. (D) Quantification of ML-SI3 effect. (E) BAPTA-AM (5 μM, 1 h pretreatment) blocked Tem-induced TFEB nuclear translocation. (F) Tem (5 μM) induced TFEB nuclear translocation in TFEB-GFP stable cells overexpressing mCherry-TRPML2. Quantification is shown in the right panel. (G) The effects of Tem (5 μM, 2 h) and ML-SA1 (5 μM, 2 h) on TFEB nuclear translocation in TFEB-GFP stable cells that were transfected with mCherry-TRPML3. Data are quantified in the left panel. mCherry-positive cells are indicated by asterisks. Scale bar = 10 μm. Data shown in B and D–G were obtained from 30 to 40 cells from at least 3 independent experiments and are presented as mean ± SEM. The individual data supporting B and D–G can be found in S1 Data. ***P < 0.001, one-way ANOVA. BAPTA-AM, 1,2-Bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid tetrakis (acetoxymethyl ester); CTRL, control; Cyt, cytoplasm; Defo, deforolimus; Eve, everolimus; GFP, green fluorescent protein; mCh, mCherry; mCherry, monomeric red fluorescent protein; ML1, TRPML1; ML-SA1, TRPML1 synthetic agonist 1; ML-SI3, TRPML1 synthetic inhibitor 3; Nuc, nuclear; O/E, overexpression; Rap, rapamycin; Seco, seco-rapamycin; Tem, temsirolimus; TFEB, transcription factor EB; ML1/TRPML1, transient receptor potential channel mucolipin 1; Zota, zotarolimus.