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. 2017 Dec 22;8:2270. doi: 10.1038/s41467-017-02332-3

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — Engagement of distinct Ca²⁺ pathways by small-molecule agonists of TFEB. a Intracellular Ca²⁺ concentration was measured in wild-type HeLa cells treated with 5 μM BAPTA-AM, 370 nM DG, 3.3 μM AD, and IKA using Fura-2-AM, and the concentration difference between compound-treated and DMSO-treated cells was normalized to the Ca²⁺ concentration in DMSO-treated cells (n = 3 independent experiments). b Confocal images of GFP-TFEB HeLa cells treated with 5 μM BAPTA-AM, 5 μM FK506, 10 μM compound C (Cmpd C) and 25 μM STO-609 together with 370 nM DG, 3.3 μM AD, and IKA for 4 h. Scale bar, 20 μm. c The graph represents the percentage of cells with GFP-TFEB translocation in b (mean ± s.d. for n = 3 independent experiments, ****p < 0.0001 by two-way ANOVA). d Representative images of GFP-TFEB HeLa cells treated with DG, AD, and IKA together with control siRNA (siLONRF1) treatment, siRNA-mediated inhibition of PPP3CB (siPPP3CB) or in combination with 5 μM calcineurin inhibitor FK506 for 4 h. Scale bar, 20 μm. e The graph represents the percentage of cells with GFP-TFEB translocation in d (mean ± s.d. for n = 3 independent experiments, ****p < 0.0001 by two-way ANOVA). Inset shows the immunoblot of HeLa cells treated with siLONRF1 or siPPP3CB. f Cell-permeable pyruvate can reverse the TFEB nuclear translocation induced by AD and IKA, but not DG. Scale bar, 20 μm. g The graph represents the percentage of cells with GFP-TFEB translocation in f (mean ± s.d. for n = 3 independent experiments, ****p < 0.0001 by two-way ANOVA)