Figure 3

The PtdIns(3,4,5)P3-mTOR signaling pathway controls transferrin uptake. (a) Schematic representation of the PtdIns(3,4,5)P3-mTOR signaling pathway. (b) Quantification of the effects of different siRNAs targeting the PtdIns(3,4,5)P3-mTOR pathway and rapamycin (RAPA) on transferrin uptake. Means ± standard error of the mean (SEM; for d-siRNAs, n = 6; for synthetic siRNAs, n = 4; for rapamycin, n = 17). (c-d) Regulation of S6 protein phosphorylation by the PtdIns(3,4,5)P3-mTOR targeting d-siRNAs. Immunofluorescent images compared the effect of mTOR and GL3 luciferase (CTR) knock-downs on phosphorylated S6 protein staining (red). Nuclei are stained with Hoechst (blue). Scale bars, 10 μm. Quantification of the staining-associated average fluorescence intensity (d). Means ± SEM (n = 6). (e) Correlation between the effect of d-siRNAs on phosphorylation of S6 protein and transferrin uptake. Means ± standard deviation (n = 2 experiments). (f) Inhibition of mTOR signaling by rapamycin (20 nM, 24 h) preferentially affects transferrin uptake when compared to DiI-labeled LDL or Alexa Fluor 488-labeled EGF. Average vesicular fluorescence intensities were measured. Mean ± SEM (n = 6 replicates).