Fig 1. MiT/TFE transcription factors regulate mTORC1 activity both in vitro and in vivo.

(A,B) Representative immuno-blotting analysis of TFEB, phospho-S6K and S6K in Tet-ON TFEB-CA cell line untreated (−DOX) or treated with Doxycycline (+DOX) for 24h. Cells were starved for a.a. for 50 min (0) and stimulated with decreasing levels (expressed as % of concentration in RPMI medium) of leucine (A) or arginine (B) for 20 min. (C) C57BL6 mice injected with a Helper-Dependent adenovirus (HDad) expressing human TFEB under the control of a liver-specific promoter (TFEB-INJ.), or with PBS (CTRL) were starved for 22h (FASTED), and then reefed for 2h (FED). Liver lysates were analyzed for levels of indicated proteins. Actin was used as loading control. The plot shows ratio of phosphorylated S6K/pan-S6K (mean of three independent experiments). (D) Immunohistochemistry analysis of liver sections from mice injected with saline PBS (CTRL) or HDAd-TFEB (TFEB-INJ.). Tissues were stained for serine 240/244 phosphorylated-S6 (P-S6). Insets show overlapping P-S6 and TFEB immunostainings in two consecutive 5μm liver sections isolated from HDad-TFEB injected mice. (E) Liver samples from mice with indicated genotypes were analyzed for the levels of S6K phosphorylation and puromycin incorporation. The plots show the ratios of phosphorylated S6K/pan-S6K and puromycin/actin expressed as relative to control mice (Tcfeb^flox/flox). (F) Phosphorylation of S6K and levels of puromycin incorporation analysis in muscle samples from mice with indicated genotypes after oral gavage of leucine. Mice were exercised where indicated. The plots show ratios of phosphorylated S6K/pan-S6K and puromycin/GAPDH. The plots in (C), (E) represent means of triplicates +/− SEM, Student t-test. The plot in (F) represent means +/− SEM; N=3; Anova (one-way) followed by Tukey’s test. In (C), (E), (F) *p < 0.05 **=p < 0.01, ***p < 0.001.