Fig. 1

Characterization of the effect of Torin1 and fresh nutrients on mTOR and endogenous TFEB. a Schematic representation of the effects of Torin1 and fresh fully-supplemented medium (FM) on the regulation of TFEB by mTOR. b Dose-response of the effect of Torin1 on mTOR activity. HeLa cells were treated with FM containing the indicated concentrations of Torin1, or kept in culture medium (non-treated, NT), for 1.5 or 3 hours, and phosphorylation of the mTOR substrates 4E-BP1 and p70-S6K1 was measured by Western blotting. c Quantification of the ratio of phosphorylated 4E-BP1 (p-4E-BP1) to total 4E-BP1. Graphs display mean values of three independent experiments normalized to NT values. Error bars denote mean ± SD of three independent experiments. Statistical significance was tested vs. NT conditions (Student’s two-tailed t-test; **, p ≤ 0.01; ***, p ≤ 0.001). d Immunofluorescence of TFEB and p-4E-BP1, as a read-out for mTOR activity, in response to FM and Torin1. HeLa cells were kept in culture medium (non-treated, NT), treated with fresh FM, or with FM supplemented with Torin1 (2 μM) for 3 hours and immunostained for TFEB and p-4E-BP1. To reveal varying intensity levels the look-up-table ‘Fire’ (ImageJ) was applied to grey scale images, representing intensity values ranging from low (dark purple) to high (white) as displayed in color scale bar. Scale bars, 20 μm