Fig. 6

Cells lacking Ate1 displayed an overactivated mTORC1/AMPk pathway. a Immunoblot analysis of p-mTORC1 (S2448), p-AMPk (T172), and LC3B levels in WT, Ate1 KO, and Ate1 KO^Ate1-1 cells after 6 h of STV or INS treatment. Ate1 and β-actin were used as Ate1 deletion and loading controls, respectively. b Quantification of p-mTOR relative to total mTOR and c p-AMPk relative to total AMPk levels. Quantifications represent the mean ± SE of three independent experiments. d Immunofluorescence of WT and Ate1 KO cells incubated in serum-rich (CONTROL) or STV conditions for 6 h. Immunostaining was performed using p-mTOR (S2448) (red), Lamp-2 (green), and Ate1 (magenta) antibodies and visualized with confocal microscopy. Nuclei (blue) were stained with Hoechst dye. Insets show higher-magnification details. e Quantification of total p-mTOR fluorescence levels. n=14 (CONTROL), n=10 (STV) Ate1 KO cells, and n=14 (CONTROL), n=9 (STV) WT cells. f Pearson's correlation coefficient (PCC) between p-mTOR and Lamp-2 signals, calculated using the "Coloc 2" plug-in of the Fiji ImageJ software. n=14 (CONTROL), n=12 (STV) Ate1 KO cells, and n=12 (CONTROL), n=9 (STV) WT cells. Statistical significance was assessed using a two-sided unpaired t test. * and # represent differences between cell types and treatments, respectively. *P≤0.05, **P≤0.01, ns=nonsignificant. Scale bar: 10 μm