Fig. 1

Inhibition of mTOR does not influence ER-to-GA VSVg trafficking in early-passage HeLa cells. a Representative time-lapse confocal images of living HeLa 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 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 (35), INK128 (45). c Representative confocal images of HeLa cells that were transfected with Str-li_VSVGwt-SBP-EGFP (green) and immunofluorescently stained for the cis-Golgi marker GM130 (magenta). Scale bar = 20 µm. d Western blot analysis of phospho-AKT (P-AKT) and phospho-S6 (P-S6) levels in protein lysates from control HeLa cells or cells after INK128 treatment (300 nM, 30 min). e Quantification of Western blot analysis of P-AKT and P-S6, normalized to tubulin, in protein lysates that were obtained from HeLa cells that were treated as in (d). **p < 0.01, ***p < 0.001 (one-sample t-test). N = 4 independent experiments