Figure 5: FRα-mediated transport of 5MTHF in Z310 cells is stimulated by monensin and transferrin.

(a) Filter-grown Z310 cells stably transfected with FRα or vector were loaded with [³H]5MTHF for 1 h. After three washing steps, cells were incubated with fresh RPMI medium containing either 7 μM monensin (Mon), 0.5 mg ml⁻¹ rat transferrin (Trans) or no supplementation. Samples from the apical and basolateral compartment were removed at the indicated time points. Subsequently, cells were lysed and radioactivity was measured in medium samples and cell lysates. Data are means±s.e.m. of three independent experiments (**P<0.01; *P<0.05; Student’s t-test). (b) Z310 cells stably transfected with FRα or vector were treated with 7 μM monensin for 22 h. The medium was collected and subjected to ultracentrifugation for exosome purification. The P100 pellets were analysed by western blotting and probed with antibodies against FRα, Flot-2 and Alix. Full-scan images are displayed in Supplementary Fig. S6. (c) To further characterize the vesicles derived from Z310 cells, the 100,000g pellet from monensin-treated Z310 cells stably expressing FRα was subjected to sucrose gradient ultracentrifugation. FRα is mainly enriched in the fractions that contained Flot-2 and Alix. Full-scan images are displayed in Supplementary Fig. S6. (d) Two-colour STED microscopy of exosomes purified from Z310 cells stably expressing FRα demonstrates a colocalization of FRα with Alix of 63%±13% (data calculated with the NIH Image J software). Scale bar, 1 μm. (e) Exosomes purified from either FRα-transfected or vector-transfected Z310 cells were stained with the dye PKH26 before they were incubated with FITC-FA for 1 h at RT. Only FRα-positive exosomes colocalize with FITC-FA and thus bind folate. Scale bar, 20 μm. (f) Apical transport of 5MTHF in primary PCPECs. Filter-grown PCPECs were loaded with 25 nM [³H]5MTHF for 24 h. Cells were then incubated with 7 μM monensin or left untreated. Radioactivity from apical medium samples was measured at the indicated time points. The increase of the apical [³H]5MTHF concentration was further enhanced by incubating the cells with monensin. Data are means±s.e.m. of two independent experiments, each done in triplicate.