Identification of a Mode I regulator of TFEB during lysosomal damage. (A) Representative fluorescence images of HeLa cells stably expressing TFEB::mNG transfected with scrambled siRNA (si Con) or siAPEX1. Cells were either untreated (control) or treated with 1 mM LLOMe for 1 h. Arrowheads indicate nuclear-localized TFEB::mNG. (B) Results of the PLA using HeLa cells stably expressing TFEB::EGFP. The indicated plasmids were transiently transfected and were either nontreated (control) or treated with 1 mM LLOMe for 1 h or for 3 h followed by the PLA procedure. (C) Representative fluorescence images of lysosomes stained with an antibody against LAMP1 (green) in WT HeLa cells or WT HeLa cells stably expressing TFEB::mNG or mNG::APEX1 under nutrient conditions. (D) Quantification of the image data shown in C (n = 3 biologically independent samples). (E) Representative immunoblots of TFEB, APEX1, and GAPDH transfected with siLuc or siAPEX1. Cells were treated with cycloheximide for the indicated times (0 h: untreated control). (F) Quantification of immunoblot data shown in E (n = 3 biologically independent samples). (G) Representative immunoblots of TFEB, mNG, and GAPDH transfected with mNG::STOP or mNG::APEX1 (KD-resistant) WT or mNG::APEX1 mutants (KD-resistant) in APEX1 KD condition. (H) Quantification of immunoblot data shown in G (n = 3 biologically independent samples). (A–C) Scale bars, 50 µm. (D–H) P values were determined by one-way ANOVA with Tukey’s multiple comparison test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001. ANOVA, analysis of variance. siLuc, siLuciferase. Source data are available for this figure: SourceData F2.
Figure 2.

Identification of a Mode I regulator of TFEB during lysosomal damage. (A) Representative fluorescence images of HeLa cells stably expressing TFEB::mNG transfected with scrambled siRNA (si Con) or siAPEX1. Cells were either untreated (control) or treated with 1 mM LLOMe for 1 h. Arrowheads indicate nuclear-localized TFEB::mNG. (B) Results of the PLA using HeLa cells stably expressing TFEB::EGFP. The indicated plasmids were transiently transfected and were either nontreated (control) or treated with 1 mM LLOMe for 1 h or for 3 h followed by the PLA procedure. (C) Representative fluorescence images of lysosomes stained with an antibody against LAMP1 (green) in WT HeLa cells or WT HeLa cells stably expressing TFEB::mNG or mNG::APEX1 under nutrient conditions. (D) Quantification of the image data shown in C (n = 3 biologically independent samples). (E) Representative immunoblots of TFEB, APEX1, and GAPDH transfected with siLuc or siAPEX1. Cells were treated with cycloheximide for the indicated times (0 h: untreated control). (F) Quantification of immunoblot data shown in E (n = 3 biologically independent samples). (G) Representative immunoblots of TFEB, mNG, and GAPDH transfected with mNG::STOP or mNG::APEX1 (KD-resistant) WT or mNG::APEX1 mutants (KD-resistant) in APEX1 KD condition. (H) Quantification of immunoblot data shown in G (n = 3 biologically independent samples). (A–C) Scale bars, 50 µm. (D–H) P values were determined by one-way ANOVA with Tukey’s multiple comparison test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001. ANOVA, analysis of variance. siLuc, siLuciferase. Source data are available for this figure: SourceData F2.

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