Additional file 6: Figure S6. of Time course decomposition of cell heterogeneity in TFEB signaling states reveals homeostatic mechanisms restricting the magnitude and duration of TFEB responses to mTOR activity modulation

Nuclear localization of TFEB is influenced by total levels of cellular TFEB. (a) HeLa cells were subjected or not to transfections with the indicated constructs and treated 24 hours post transfection with 2 μM Torin1 for 3 hours or left non-treated (NT). Representative images demonstrate the subcellular distribution of TFEB fluorescence for endogenous TFEB (TFEB immunofluorescence, IF), or transiently overexpressed GFP-TFEB, expressed alone or coexpressed with RFP-tagged 14-3-3 protein isoform YWHAG. The look-up-table ‘Fire’ (ImageJ) was applied to grey scale images of TFEB or GFP-TFEB fluorescence, representing ranging from high (white) to low (dark purple) intensity values, as displayed in color scale bar. Scale bars (white line), 20 μm. (b) Quantification of the number of cells with mainly nuclear TFEB fluorescence. At least 30 cells were scored per condition and experiment in three independent experiments. Statistical significance was tested using two-tailed Student’s t-test (**, p ≤ 0.01; ***, p ≤ 0.001). (JPG 1702 kb)