Figure 5.
S466 S467 S469 phosphorylation is required for TFEB activity. (A) Immunoblot of protein lysates of MEFs overexpressing WT TFEB-GFP and TFEB-GFPS466A,S467A,S469A incubated in complete media (Control) or in presence of AICAR (2 mM) or Torin1 (1 μM) for 2 h. Data are representative of three independent experiments. (B) Representative images of MEFs overexpressing WT TFEB-GFP and TFEB-GFPS466A,S467A,S469A upon treatment as indicated in (A). 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 overexpressing WT TFEB-GFP and TFEB-GFPS466A,S467A,S469A with nuclear TFEB-GFP upon treatments as indicated in (A) (mean ± SEM of three independent experiments, two-way ANOVA, ns = not significant, ****P < 0.0001; n > 200 cells per condition). (D) Representative images of MEFs expressing WT TFEB-GFP and TFEB-GFPS466A,S467A,S469A after 1 h of incubation with DQ-BSA-Red followed by a 2 h chase in complete media in presence of DMSO (Control), AICAR (2 mM) or Torin1 (1 μM) 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 overexpressing WT TFEB-GFP and TFEB-GFPS466A,S467A,S469A upon treatment as indicated in (D) (mean ± SEM of four independent experiments, two-way ANOVA, ns = not significant, *P < 0.05, **P < 0.01; n > 200 cells per condition). (F) Relative Tfeb and Tfe3 transcriptional activity, as determined by CLEAR-luciferase promoter activity normalized against CMV-Renilla in MEFs expressing WT TFEB-GFP and TFEB-GFPS466A,S467A,S469A upon treatment as indicated in (A) (mean ± SEM of the luminescence fold change from four independent experiments, two-way ANOVA, ns = not significant, **P < 0.01). (G) Quantification of the number of LAMP1 puncta in MEFs overexpressing WT TFEB-GFP and TFEB-GFPS466A,S467A,S469A upon treatment as indicated in (A) (mean ± SEM of three independent experiments, two-way ANOVA, ns = not significant, *P < 0.05; **P < 0.01; n > 50 cells per condition). (H-I) Relative quantitative real‐time PCR analysis of Tfeb and Tfe3 target genes mRNA transcript levels in MEFs expressing WT TFEB-GFP and TFEB-GFPS466A,S467A,S469A upon treatment as indicated in (A) (mean ± SEM of the RNA fold change of indicated mRNAs from four independent experiments, two-way ANOVA, ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001)