Automated image-based quantification of iron-loaded transferrin uptake in HeLa cells. (a) Schematic representation of the transferrin-mediated iron uptake system. Transferrin receptors (TFRC, grey ovals) cycle between the plasma membrane (PM) and the endosomal recycling compartment (ERC) using vesicular carrier (black circles). Iron (Fe3+, blue circles) binds to TFRC at the cell surface and is released into the ERC. At the steady-state, the quantity of internalized transferrin depends on the rate of transferrin endocytosis (kendo), the rate of transferrin recycling (kexo), and on the total number of cycling transferrin receptors. (b) Fluorescent transferrin uptake in mock-transfected HeLa cells (red). Hoechst-stained nuclei (blue) were used to segment the images and create the perinuclear regions (yellow) used to measure the fluorescence intensity in the 'red' channel. (c) Histogram of single cell fluorescence intensities (the median (F) is the transferrin uptake index used in this study). (d) Time-course of fluorescent transferrin uptake (red circles) and recycling (blue circles). The dashed line indicates the time point chosen for the screen. Means ± standard error of the mean (n = 4 replicates). (e) Images and quantification of fluorescent transferrin uptake in cells transfected with d-siRNAs targeting GL3 luciferase (CTR), the μ2 subunit of the AP2 adapter (AP2M1) or the clathrin heavy chain (CLTC). Means ± standard error of the mean (n = 3 experiments). Scale bars, 10 μm.