Fig. 7

Effect of ERK, proteasome and transcriptional inhibition on mTOR inhibition-mediated TFEB activation. a HeLa cells were treated with fresh FM supplemented with U0126 (10 μM) alone or in co-treatment with Torin1 (2 μM), or subjected to nutrient deprivation (ND) alone or in combination with U0126. Following immunostaining for TFEB and labeling of nuclei with Hoechst 33342 cells were analyzed by multispectral imaging cytometry. For visualization, the data for FM supplemented with U0126 is included both for Torin1 (left) and for ND (right) treatments. Bars report mean subpopulation distributions among three independent experiments ± SD. Statistical significance was tested based on the “Active” subpopulation vs. non-treated control (NT), unless otherwise indicated by horizontal lines (Student’s two-tailed t-test; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; n.s., p > 0.05). b Mean population response of TFEB concentration in HeLa cells treated as in (a), shown relative to the non-treated levels (NT). Unless indicated by horizontal lines, statistical significance was tested vs. non-treated control, NT (Student’s two-tailed t-test; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; n.s., p > 0.05). c Mean population response of TFEB concentration (relative to the non-treated levels, NT) in HeLa cells treated with Epox (1 μM), ActD (1 μg/mL) or vehicle control (DMSO) alone or in combination with Torin1 (2 μM). Bars report the mean among four independent experiments ± SD. Statistical significances were tested with Student’s two-tailed t-test (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; n.s., non-significant). d Mean population response of TFEB subcellular localization and subpopulation distributions for HeLa cells treated as in (c). Bars report the mean among four independent experiments ± SD. Statistical significances were tested with Student’s two-tailed t-test (**, p ≤ 0.01; ***, p ≤ 0.001; n.s., non-significant). Subpopulations were compared based on the “Active” phenotype. e Mean TFEB concentration for the three activation phenotypes in a subset of cells treated with Torin1 alone or in co-treatment with epoxomicin, as indicated in (c). Bars represent the mean among all cells in each cluster for the indicated treatments ± SD