Fig. 7
B12 gets rapidly internalized via clathrin-coated pits and traffics into early endosomes and lysosomes. a HPB-ALL cells were treated with 125 nm (18 μg/mL) B12, for the indicated time points at 37 °C and surface-bound B12 was analyzed by flow cytometry, as described in “Materials and methods”. Relative signal of B12 bound to IL-7Rα at the cell surface for each time point was calculated as the geometric mean intensity of fluorescence normalized to time 0. A commercially available anti-IL-7Rα from R&D was used for comparison. b HPB-ALL cells were cultured in the presence or absence of IL-7 (50 ng/mL) with B12, for the indicated time points. a, b Data represent the mean from at least two independent experiments. c Analysis of B12 colocalization with clathrin was performed by confocal microscopy. HPB-ALL cells were plated on poly-d-lysine coverslips, incubated with B12 at 4 °C for 45 min and subsequently with secondary anti-human IgG Alexa 647 (red). After the incubation with B12, cells were shifted to 37 °C to let the internalization happen. Clathrin-coated pits were detected after 30 min of incubation, by permeabilizing the cells and staining with anti-clathrin-alexa 568 (green) while the nuclei was stained with DAPI. Images were acquired using LSM 880 and the representative picture is a maximum intensity projection of a Z-stack image, 63× objective. For analysis, at least 10 fields of images were analyzed, each image having ~ 100 cells in the frame. Percent of cells with internalization were analyzed by normalizing the cells with colocalized (yellow) spots to the total cells having both the fluorophores. d B12 localization in early endosomes was detected after 30 min of incubation at 37 °C, using an anti-EEA-1-alexa 555 antibody, and trafficking to lysosomes after 60 min e was detected using an anti-LAMP-2-alexa 488 antibody. Statistical analysis was performed using unpaired t test (***p < 0.001, **p < 0.01, *p < 0.05)
