Fig. 6
Single cell correlation of nutrient deprivation-induced TFEB activity and downstream lysosomal response by multispectral imaging cytometry. 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 labelled with Hoechst 33342. Following, cells were analyzed by ISX multispectral imaging cytometry. a Bright field and fluorescence images of representative cells for the indicated treatments. b Time course of the mean population response of LAMP1 protein levels, shown relative to time point ‘0’. Reported values represent the mean among three independent experiments ± SD. 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; ***, p ≤ 0.001). c Mean LAMP1 concentration for the three TFEB activation phenotypes. 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 (Student’s two-tailed t-test; ***, p ≤ 0.001; n.s. p > 0.05). d Representative fluorescence images for different ranges of the feature “LAMP1 Max Contour Position” to assess lysosomal positioning. e Time course of the mean population response of “LAMP1 Max Contour Position”, shown relative to time point ‘0’. Reported values represent the mean among three independent experiments ± SD. 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). f Mean “LAMP1 Max Contour Position” for the three TFEB activation phenotypes. 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). g Percentage of cells with values of “LAMP1 Max Contour Position” within the ranges specified in (d), presented separately for each TFEB activation phenotype