Figure 2.

BBBECs transport⁵⁵Fe -Tf and⁵⁵Fe -FTH1 via exosomes to the basal chamber. ECs grown on the 0.4 μm pore size filters were incubated with GW4869 20 μM or 1 mg/ml of ⁵⁵Fe-Tf or 100 μg/ml of ⁵⁵Fe-FTH1 added to the apical chamber. After 24 h, exosomes were isolated from the basal chamber. The medium was measured for ⁵⁵Fe activity in the exosomes versus supernatant fractions. ⁵⁵Fe activity was measured in a liquid scintillation counter, results are expressed in DPM/ml. A, compares the ⁵⁵Fe-Tf activity in the exosomes and supernatant fractions. The data shown are the mean ± S.D. of three independent replicates, Unpaired t test, ∗∗p < 0.01. B, compares the ⁵⁵Fe-His-FTH1 activity in the exosomes and supernatant fractions. The data shown represent the mean ± S.D. of three independent replicates, Unpaired t test. ∗∗p < 0.01. C and D, demonstrate the corresponding immunoblot analysis for Tf and His-FTH1 in the respective exosomes (Exo) and supernatant (Sup) fractions. E, illustrates the exosome concentration released in control versus GW4869-treated cells. F, the immunoblots of CD81 and CD63 in control versus GW4869-treated cells. G and H, illustrate the exosomal ⁵⁵Fe-Tf and ⁵⁵Fe-His-FTH1 activity respectively in control versus GW4869-treated cells. The data shown are the mean ± S.D. of three independent replicates, Unpaired t test, ∗p < 0.05. All experiments were performed with three technical replicates in each biological replicate. BBBECs, blood–brain barrier endothelial cells; ECs, endothelial cells; FTH1, H-ferritin; Tf, transferrin.