Fig. 6

Src regulates cell growth, TFEB localization and autophagy. a, b The histogram shows the cell size of SH-SY5Y (a) and MEF (b) cells treated with DMSO or PP2 (5 μM for 24 h). The bar diagram represents the mean diameter of at least 10⁴ cells. Error bars represent values in ± SEM. ***p < 1 × 10⁻⁶. c SH-SY5Y cells expressing GFP-LC3 were starved and treated with vehicle (DMSO), PP2 (10 μM), or rapamycin (600 nM) for the last 2 h of starvation and then stimulated with amino acids (30 min). Box plots represent quantified data of lipidated-LC3 puncta from at least 35 cells. *p < 0.05 and ***p < 0.001. Bar indicates 20 μm. d Immunoblot analysis of SH-SY5Y cells transiently transfected with scrambled shRNA or shSrc prior to starvation and amino acid stimulation (30 min). e Immunoblot analysis of SH-SY5Y cells left in growth media or starved and treated with vehicle (DMSO) or PP2 (10 μM) for the last 2 h of starvation and then stimulated with amino acids (30 min). Box plots in d and e represent SE of n = 3 independent experiments. *p < 0.05 and **p < 0.01. f HeLa cells, stably transfected with Flag-TFEB, were transiently transfected with scrambled or shSrc and treated as in d. Cells were immunostained with Flag (green) for the analyses of cytosolic and nuclear localization of TFEB. Bar indicates 80 μm. g HeLa cells, stably transfected with Flag-TFEB, were treated as in e. Torin1 (300 nM for 2 h) was used as a control (mTOR inhibitor). Cells were immunostained with Flag (green) for the analyses of cytosolic and nuclear localization of TFEB. Bar indicates 40 μm. h HeLa cells, stably transfected with Flag-TFEB, were treated as in e prior to immunofluorescence labeling of LAMP2 (red) and Flag (green). Representative cells are shown where yellow or orange pixels indicate co-localization in the merged images. In all images, insets show selected fields that were magnified four times and their overlays. Bar indicates 30 μm. GAPDH was used as a loading control in all immunoblot assays. Statistical differences between groups in a–e were determined using ANOVA with Tukey’s post hoc test