Fig. 3

Multispectral imaging cytometry quantification of TFEB localization and levels in response to mTOR activity modulations. Cells were kept in culture medium (NT, non-treated), or treated with fresh FM or FM supplemented with Torin1 (2 μM). Following, cells were immunostained for TFEB and nuclei labelled with Hoechst 33342. a Representative fluorescence images and quantified TFEB nuclear localization in HeLa cells at 3 hours of treatment, measured with high-resolution wide field imaging (WF, left panels) or with the multispectral imaging cytometer ImageStreamX (ISX, right panels). Graphed values represent the mean ± SD nuclear/cytoplasmic ratio of 25 to 30 randomly selected cells of one representative experiment from three independent repetitions. Statistical significances were tested vs. NT control (Student’s two-tailed t-test; ***, p ≤ 0.001; n.s., p > 0.05). b-e Time course of mean population response of TFEB subcellular localization and protein levels for treatments with Torin1 or fresh FM, in HeLa and MCF7 cell lines. Concentrations are shown relative to time point ‘0’. Reported values represent the mean among three independent experiments ± SD. Statistical significances were tested vs. time point ‘0’, which corresponds to the NT control (Student’s two-tailed t-test; *, p ≤ 0.05; **, p ≤ 0.01)