Figure 6.
S466A S467A S469A blocks MTORC1-regulated TFEB activity. (A) Immunoblot of protein lysates of MEFs expressing WT TFEB-GFP, TFEB-GFPS466A,S467A,S469A, TFEB-GFPS142A,S211A (TFEB-GFP[CA]) or TFEB-GFP[CA]S466A,S467A,S469A incubated in complete media. Data are representative of three independent experiments. (B) Representative images of indicated MEF lines incubated in complete media. Cells were fixed and the GFP fluorescence directly observed by microscopy. Scale bar: 20 μm. Images are representative of three independent experiments. (C) Quantification of the percentage of MEFs with nuclear TFEB-GFP in complete media (mean ± SEM of three independent experiments, one-way ANOVA, ns = not significant, ****P < 0.0001; n > 200 cells per condition). (D) Representative images of MEFs after 1 h of incubation with DQ-BSA-Red followed by a 2 h chase in complete media prior to fixation. Scale bar: 20 μm. Images are representative of four independent experiments. (E) Relative lysosomal activity, as determined by DQ-BSA assay, in MEFs upon treatment as indicated in (D) (mean ± SEM of four independent experiments, one-way ANOVA, ns = not significant, **P < 0.01; n > 200 cells per condition). (F) Quantification of the number of LAMP1 puncta in MEFs overexpressing WT TFEB-GFP, TFEB-GFPS466A,S467A,S469A, TFEB-GFPS142A,S211A (TFEB-GFP[CA]) or TFEB-GFP[CA]S466A,S467A,S469A incubated in complete media (mean ± SEM of three independent experiments, two-way ANOVA, ns = not significant, **P < 0.01; ***P < 0.001; n > 50 cells per condition). (G) Relative Tfeb and Tfe3 transcriptional activity, as determined by CLEAR-luciferase promoter activity normalized against CMV-Renilla in MEFs were incubated in complete media (mean ± SEM of the luminescence fold change from five independent experiments, one-way ANOVA, ns = not significant, ***P < 0.001). (H) Relative quantitative real‐time PCR analysis of TFEB and TFE3 target genes mRNA transcript levels in MEFs were incubated in complete media (mean ± SEM of the RNA fold change of indicated mRNAs from four independent experiments, two-way ANOVA, **P < 0.01, ***P < 0.001, ****P < 0.0001)