APEX1 is required for TFEB expression and stability. (A) Quantification of the relative expression of TFEB, TFE3, and MITF by qPCR in HeLa cells transfected with siLuc or siAPEX1 under nutrient conditions (n = 3 biologically independent samples). (B) Representative immunoblots of TFEB, p-TFEB (Ser211), APEX1, and GAPDH in WT HeLa cells transfected with EGFP stop (negative control) or EGFP::APEX1, either untreated (control) or treated with 1 mM LLOMe for 1 h. (C) Quantification of 2×CLEAR (TFEB RE)-luciferase reporter assays in HeLa cells stably expressing mNG or APEX1::mNG transfected under 1 mM LLOMe treatment for 3-h conditions (n = 3 biologically independent samples). (D) Representative immunoblots of TFEB, APEX1, and GAPDH in WT HeLa cells that were either untreated (control) or treated with MG132 for 3 h. Cells were transfected with siLuc or siAPEX1. (E) Quantification of the image data shown in D (n = 3 biologically independent samples). (F) Representative immunoblots of TFEB and GAPDH in WT HeLa cells treated with chloroquine for 4 h. Cells were transfected with siAPEX1. (G) Quantification of image data shown in F (n = 3 biologically independent samples). (H) Representative immunofluorescence images of HeLa cells stained using an antibody against endogenous γH2AX (green). Cells were transfected with siLuc or siAPEX1. (I) Representative immunoblots of γH2AX and GAPDH in WT HeLa cells that were transfected with siLuc or siAPEX1. (J) Quantification of image data shown in I (n = 3 biologically independent samples). (K) Schematic representation of APEX1 mutant constructs. (L) Representative fluorescence images of HeLa cells stably expressing mNG::APEX1 WT or mNG::APEX1 mutants. (M) Results of the PLA using WT HeLa cells stably expressing mNG::stop, mNG::APEX1 WT, or mNG::APEX1 mutants. Cells were transfected with siAPEX1. The indicated plasmids were transiently transfected and were either untreated (control) or treated with 1 mM LLOMe for 1 h followed by the PLA procedure. (H, L, and M) Scale bars, 50 μm. (E) P values were determined by ANOVA with Tukey’s multiple comparison test; *P < 0.05. (A and C) P values were determined by a t test; *P < 0.05, **P < 0.01. Source data are available for this figure: SourceData FS4.
Figure S4.

APEX1 is required for TFEB expression and stability. (A) Quantification of the relative expression of TFEB, TFE3, and MITF by qPCR in HeLa cells transfected with siLuc or siAPEX1 under nutrient conditions (n = 3 biologically independent samples). (B) Representative immunoblots of TFEB, p-TFEB (Ser211), APEX1, and GAPDH in WT HeLa cells transfected with EGFP stop (negative control) or EGFP::APEX1, either untreated (control) or treated with 1 mM LLOMe for 1 h. (C) Quantification of 2×CLEAR (TFEB RE)-luciferase reporter assays in HeLa cells stably expressing mNG or APEX1::mNG transfected under 1 mM LLOMe treatment for 3-h conditions (n = 3 biologically independent samples). (D) Representative immunoblots of TFEB, APEX1, and GAPDH in WT HeLa cells that were either untreated (control) or treated with MG132 for 3 h. Cells were transfected with siLuc or siAPEX1. (E) Quantification of the image data shown in D (n = 3 biologically independent samples). (F) Representative immunoblots of TFEB and GAPDH in WT HeLa cells treated with chloroquine for 4 h. Cells were transfected with siAPEX1. (G) Quantification of image data shown in F (n = 3 biologically independent samples). (H) Representative immunofluorescence images of HeLa cells stained using an antibody against endogenous γH2AX (green). Cells were transfected with siLuc or siAPEX1. (I) Representative immunoblots of γH2AX and GAPDH in WT HeLa cells that were transfected with siLuc or siAPEX1. (J) Quantification of image data shown in I (n = 3 biologically independent samples). (K) Schematic representation of APEX1 mutant constructs. (L) Representative fluorescence images of HeLa cells stably expressing mNG::APEX1 WT or mNG::APEX1 mutants. (M) Results of the PLA using WT HeLa cells stably expressing mNG::stop, mNG::APEX1 WT, or mNG::APEX1 mutants. Cells were transfected with siAPEX1. The indicated plasmids were transiently transfected and were either untreated (control) or treated with 1 mM LLOMe for 1 h followed by the PLA procedure. (H, L, and M) Scale bars, 50 μm. (E) P values were determined by ANOVA with Tukey’s multiple comparison test; *P < 0.05. (A and C) P values were determined by a t test; *P < 0.05, **P < 0.01. Source data are available for this figure: SourceData FS4.

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