Fig. 4
Clustering-based analysis of subpopulation dynamics of TFEB activity in response to mTOR activity modulation by fresh FM and Torin1. Subpopulation analysis of ISX multispectral imaging cytometry datasets from Fig. 3. a Schematic representation of the analysis workflow. Extracted feature values from all treatments and time points were merged and analyzed using SPADE software for the identification of phenotypically similar clusters. Clustering results were iteratively checked until finding a combination of features and number of clusters that yielded biologically sound and reproducible results. Finally, evolution of subpopulations was observed by tracing the percentage of cells belonging to each cluster for each treatment and time point. b Cluster phenotypes in HeLa cells. Bars represent the mean among all cells in each cluster ± SD (including FM, Torin1, and all repetitions and time points). The number of cells equals 48774, 42994 and 23793 for cluster 1, 2 and 3, respectively. Statistical significances were tested between clusters on 1000 randomly selected cells (two-sided Wilcoxon-rank-sum test; ***, p ≤ 0.001). c Cumulative frequency distribution for selected features that were excluded during the generation of the clusters. Bars on the top right corners display the mean value among all cells in each cluster ± SD. Statistical significances were tested between clusters on 1000 randomly selected cells (Student’s two-tailed t-test; ***, p ≤ 0.001). d Clusters phenotype in MCF7 cells. Bars represent the mean among all cells in each cluster ± SD. Statistical significances were tested between clusters on 1000 randomly selected cells (two-sided Wilcoxon-rank-sum test; ***, p ≤ 0.001). e Mean TFEB protein levels for the three clusters. Bars represent the mean among all cells in each cluster ± SD. Statistical significances were tested between clusters on 1000 randomly selected cells (Student’s two-tailed t-test; ***, p ≤ 0.001). f-g Subpopulation dynamics for the indicated treatments and cell lines. Reported values represent the mean among three independent experiments ± SD. Regions shaded in grey highlight different stages in TFEB dynamic response. R1: short-term rheostatic response, R2: long-term response