Fig. 5

Mean population and subpopulation response of TFEB to nutrient deprivation. HeLa cells were kept in culture medium (NT, non-treated) or subjected to nutrient deprivation. At the indicated time points, cells were immunostained for TFEB and LAMP1, and Nuclei were labelled with Hoechst 33342. Following, cells were analyzed by ISX multispectral imaging cytometry. a Time course of the mean population response of TFEB subcellular localization and protein levels. Concentrations are shown relative to time point ‘0’. Reported values represent the mean among three independent experiments ± SD. Unless specified by horizontal lines, statistical significance was tested vs. time point ‘0’, which corresponds to the NT control (Student’s two-tailed t-test; *, p ≤ 0.05; **, p ≤ 0.01). b Mean TFEB subcellular localization and protein levels for the three activation phenotypes in a cell population subjected to nutrient deprivation. Bars represent the mean among all cells in each cluster ± SD (from all repetitions and time points). Statistical significance was tested between clusters on 1000 randomly selected cells (two-sided Wilcoxon-rank-sum test; ***, p ≤ 0.001). c Dynamics of the distribution of cells among the three activation phenotypes in a cell population subjected to nutrient deprivation. Reported values represent the mean among three independent experiments ± SD. Shaded regions highlight different stages in TFEB dynamic response. R1: first activation wave, R2: re-inactivation, R3: second activation wave