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. 2021 Jul 27;18(1):204–222. doi: 10.1080/15548627.2021.1954779

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This work was authored as part of the Contributor’s official duties as an Employee of the United States Government and is therefore a work of the United States Government. In accordance with 17 U.S.C. 105, no copyright protection is available for such works under U.S. Law.

This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.

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Figure 7. — LRRK2^G2019S-mediated TFEB nuclear translocation requires Ca²⁺, NAADP, and TPCN2 (A) Confocal imaging in HeLa cells expressing TFEB-mCherry and a control vector (MT) or His-LRRK2^G2019S with or without BAPTA-AM (fast calcium chelator, 10 μM, 1 h), EGTA-AM (slow calcium chelator, 10 μM, 1 h), FK506 (calcineurin inhibitor, 5 nM, 1 h), control siRNA, PPP3CB siRNA, or MCOLN1 siRNA. Scale bar: 10 μm. (B) Quantification TFEB nuclear translocation from (A) (n = 3, ≥ 50 cells/repeat). (C) Confocal imaging in HeLa cells expressing TFEB-mCherry and a control vector (MT), or TFEB-mCherry and His-LRRK2^G2019S with or without Ned-19 (NAADP inhibitor, 100 μM, 1 h), PERK inhibitor (PERK-I, 10 μM, 1 h), control siRNA, or TPCN2 siRNA. Scale bar: 10 μm. (D) Quantification of TFEB nuclear translocation from (C) (n = 3, ≥ 50 cells/repeat). (E) Immunoblot of endogenous TFEB from nuclear fractions of HEK293T cells transfected with a control vector or His-LRRK2^G2019S (GS) and either control siRNA or PPP3CB siRNA (PPP3CB KD). (F) Immunoblot of endogenous TFEB from nuclear fractions in HEK293T cells transfected with a control vector or His-LRRK2^G2019S (GS) and treated with Ned-19 (100 μm, 2 h). (G) Confocal imaging in HeLa cells transfected with TFEB-mCherry and TPC2-EGFP. DAPI and TFEB-mCherry colocalization channel is shown on the right. Scale bar: 10 μm. (H) Quantification of % cells exhibiting TFEB nuclear translocation from (G) (n = 3, ≥ 50 cells/repeat). (I) Immunoblotting of endogenous TFEB following nuclear fractionation of HEK293T cells expressing TPCN2-EGFP. For western data, histone H3 and GAPDH are shown as fractionation and loading controls. Western bands are quantified, normalized to the loading control, and presented relative to control lane. (*p < 0.05; **p < 0.002, ***p < 0.0001, one-way ANOVA with post hoc Tukey’s HSD).