Fig. 2

Inhibition of mTOR affects ER-to-GA VSVg trafficking in PC12 cells. a Representative time-lapse confocal images of living PC12 cells that were transfected with Str-li_VSVGwt-SBP-EGFP and treated with INK128 (300 nM, 30 min) or untreated (control). Trafficking of VSVg-EGFP was visualized using the RUSH assay for 60 min after the addition of biotin (time 0). Scale bar = 20 µm. b Quantitative analysis of the experiments performed as in (a). The graph depicts VSVg-EGFP fluorescence intensity in the Golgi apparatus region at each time point, normalized to the maximum value. The data are expressed as the mean for all of the analyzed cells. Error bars indicate SEM. N = 4 independent experiments. Number of cells per variant (n): Untreated (38), INK128 (27). c Comparison of VSVg-EGFP fluorescence intensity in the Golgi apparatus region at 16 min (left panel) and 34 min (right panel). The data are expressed as the mean for all of the analyzed cells. Error bars indicate SEM. *p < 0.05, ***p < 0.001 (Mann–Whitney test). The number of independent experiments and analyzed cells per variant are the same as in (b). d Representative confocal images of PC12 cells that were transfected with Str-li_VSVGwt-SBP-EGFP (green) and immunofluorescently stained for the cis-Golgi marker GM130 (magenta). Scale bar = 5 µm. e Western blot analysis of phospho-AKT (P-AKT) and phospho-S6 (P-S6) levels in protein lysates from control PC12 cells or cells after INK128 treatment (300 nM, 30 min). f Quantification of Western blot analysis of P-AKT and P-S6, normalized to tubulin, in protein lysates that were obtained from PC12 cells that were treated as in (e). ***p < 0.001 (one-sample t-test). N = 3 independent experiments