Transferrin uptake assay

For the transferrin internalization assay, HeLa cells in a 24-well plate were transfected with siRNA specific for RME-6 or control siRNA. At 74 h post-transfection, cells were starved for 4 h in CO₂-independent DMEM containing 0.2% BSA and then incubated with biotinylated and ruthenium-labeled transferrin at 10 μg/ml, also in CO₂-independent DMEM containing 0.2% BSA for the indicated time points. Labeling of biotinylated transferrin with the SULFO-TAG-NHS-ester [ruthenium (II) tris-bipyridine, N-hydroxysuccinimide; Meso Scale Discovery (MSD)] was performed as suggested by the manufacturer (MSD). After incubation, the cells were placed on ice and the transferrin solution was discarded. Cells were immediately washed with ice cold PBS containing 1 mM CaCl₂ and 0.5 mM MgCl_2, and then washed with DMEM containing 0.2% BSA, pH 2.8, for 2 min (alternatively, 0.5 M NaCl and 0.2 M acetic acid, pH 2.8, were used). Cells were washed again with 1 × PBS and lysed in ice cold lysis buffer (50 mM Tris/HCl, 50 mM NaCl, 1% Triton X-100, 1 × Roche Complete proteinase inhibitor). For lysis, plates were incubated on ice for 5 min and transferred to − 80 °C overnight or for 15 min. After thawing the plates, the lysates were transferred to a 1.5 ml Eppendorf tube, aspirated several times, centrifuged at 13,000×g for 10 min at 4 °C, and the cleared supernatant was saved. Three times 40 μl aliquots of each cell lysate containing the internalized biotinylated and ruthenium-labeled transferrin were added to individual wells of a 96-well MSD-High-Bind Multi-Array plate, with carbon electrodes coated with streptavidin, which were previously blocked with 3% MSD Blocker A in MSD Tris wash buffer. The MSD plate was incubated for 1 h at room temperature with agitation. Finally, the cell lysates were discarded, wells were washed once with MSD read buffer T containing surfactant, and the plates were read immediately on a SECTOR Imager 6000 (Meso Scale Discovery). The amount of internalized transferrin was standardized in relation to the total protein concentration of the lysate and expressed as percent of the amount of internalized transferrin in control cells, at the different time points, or comparative relative units.

The mean of three biological repeats, each measured in three individual wells, was calculated and the ± SEM is shown. Since this assay was highly reproducible, error bars are not visible.