Identification of Mode II regulators of TFEB during lysosomal damage. (A) Representative fluorescence images of ATG7 KO cells stably expressing TFEB::mNG and transfected with scrambled siRNA (siCon), siCCT7, and siTRIP6. Cells were either untreated (control) or treated with 1 mM LLOMe for 3 h. Arrowheads indicate nuclear-localized TFEB::mNG. (B and C) Representative immunoblots of FLAG and TFEB in WT or ATG7 KO HeLa cells with or without stable TFEB::mNG expression. FLAG::CCT7 (B) or FLAG::TRIP6 (C) was transiently transfected and then treated or not treated (control) with 1 mM LLOMe for 3 h, followed by immunoprecipitation with an antibody against mNG. (D and F) Representative fluorescence images of HeLa cells stably expressing mNG::CCT7 (D) or mNG::TRIP6 (F) (green). Lysosomes were stained with an antibody against LAMP1 (magenta). Cells were either treated or not treated (control) with 1 mM LLOMe for 3 h. (E and G) Quantification of immunofluorescence data shown in D or G, respectively (n = 3 biologically independent samples). (H) Representative immunoblots of TFEB, p-TFEB (Ser211), and tubulin under control conditions and 1 mM LLOMe treatment for 3 h in WT and ATG7 KO HeLa cells. Cells were transfected with siLuc, siCCT7, and siTRIP6. (I) Quantification of image data of p-TFEB (Ser211)/tubulin shown in H (n = 3 biologically independent samples). (J) Representative immunoblots of TFEB, p-TFEB (Ser211), CCT7, TRIP6, and GAPDH in WT HeLa cells under control and 3 or 18 h after 1 mM LLOMe treatment conditions. Cells were transfected with siLuc, siCCT7, and siTRIP6. (A, D, and F) Scale bars, 50 µm. (E and G) P values were determined by a t test; **P < 0.01. (I) P values were determined by ANOVA with Tukey’s multiple comparison test; **P < 0.01, ***P < 0.005. Source data are available for this figure: SourceData F3.
Figure 3.

Identification of Mode II regulators of TFEB during lysosomal damage. (A) Representative fluorescence images of ATG7 KO cells stably expressing TFEB::mNG and transfected with scrambled siRNA (siCon), siCCT7, and siTRIP6. Cells were either untreated (control) or treated with 1 mM LLOMe for 3 h. Arrowheads indicate nuclear-localized TFEB::mNG. (B and C) Representative immunoblots of FLAG and TFEB in WT or ATG7 KO HeLa cells with or without stable TFEB::mNG expression. FLAG::CCT7 (B) or FLAG::TRIP6 (C) was transiently transfected and then treated or not treated (control) with 1 mM LLOMe for 3 h, followed by immunoprecipitation with an antibody against mNG. (D and F) Representative fluorescence images of HeLa cells stably expressing mNG::CCT7 (D) or mNG::TRIP6 (F) (green). Lysosomes were stained with an antibody against LAMP1 (magenta). Cells were either treated or not treated (control) with 1 mM LLOMe for 3 h. (E and G) Quantification of immunofluorescence data shown in D or G, respectively (n = 3 biologically independent samples). (H) Representative immunoblots of TFEB, p-TFEB (Ser211), and tubulin under control conditions and 1 mM LLOMe treatment for 3 h in WT and ATG7 KO HeLa cells. Cells were transfected with siLuc, siCCT7, and siTRIP6. (I) Quantification of image data of p-TFEB (Ser211)/tubulin shown in H (n = 3 biologically independent samples). (J) Representative immunoblots of TFEB, p-TFEB (Ser211), CCT7, TRIP6, and GAPDH in WT HeLa cells under control and 3 or 18 h after 1 mM LLOMe treatment conditions. Cells were transfected with siLuc, siCCT7, and siTRIP6. (A, D, and F) Scale bars, 50 µm. (E and G) P values were determined by a t test; **P < 0.01. (I) P values were determined by ANOVA with Tukey’s multiple comparison test; **P < 0.01, ***P < 0.005. Source data are available for this figure: SourceData F3.

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