Figure 4From: siRNA screen of the human signaling proteome identifies the PtdIns(3,4,5)P3-mTOR signaling pathway as a primary regulator of transferrin uptakemTOR controls transferrin uptake by regulation of the number of transferrin receptors per vesicle and not endocytosis or recycling rates. (a) Effect of rapamycin on the time-course of fluorescent transferrin endocytosis (left) and recycling (right). Means ± standard error of the mean (SEM; n = 4 replicates). (b) Lack of effect of rapamycin on the rate constants for endocytosis and recycling. The rate constants, tau-1, were calculated from a single exponential fit of the time-course data. (c) Effect of the PtdIns(3,4,5)P3-mTOR-targeting d-siRNAs on the number of transferrin receptors (TFRC) per cell (measured by immunofluorescence on permeabilized cells). Means ± SEM (n = 4). (d) Correlation between transferrin uptake (F, vertical axis) and transferrin receptor numbers (immunofluorescence measurements on permeabilized cells, horizontal axis) in HeLa cells in which PtdIns(3,4,5)P3-mTOR regulators were silenced. Means ± SEM (n = 4). (e) Selective effect of rapamycin on transferrin receptor staining (TFRC) compared to tubulin and actin staining. Means ± SEM (n = 6 replicates). (f) Rapamycin preferentially reduces the number of transferrin molecules per vesicles. Histogram of the relative transferrin fluorescence intensity per vesicle for control (CTR) and rapamycin (RAPA)-treated cells. Means ± SEM (n = 6 replicates). Inset shows the average intensity measured for the 30 brightest vesicles per cell. (g) Rapamycin has only a minor effect on the number of endocytic vesicles. Bar diagram shows means ± SEM (n = 6 replicates).Back to article page