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. 2023 Jan 3;299(2):102868. doi: 10.1016/j.jbc.2022.102868

Figure 4.

Figure 4

BBBECs iron concentration alters the release of exosomes and exosomes containing FTH1 and Tf into the basal chamber. ECs were grown on the 0.4 μm pore size filters treated with ferric ammonium citrate (FAC, 100 μmol/L) or deferoxamine (DFO, 100 μmol/L) for 24 to 48 h, the basal media were collected and the exosomes isolated. This experiment was designed to demonstrate exosomes release, and the Tf and FTH1 content of the exosomes were impacted by the BBBECs iron status. A, illustrates the immunoblot analysis of exosomes for detection of FTH1, transferrin, CD63, and CD81 expression. B, the corresponding graphical analysis of FTH1, transferrin, CD63, and CD81/ponceau S expressions are presented as fold change relative to the control (data have been normalized to the untreated control for each independent experiment). The data shown represent the mean ± S.D. of three independent replicates and analyzed by one way-ANOVA, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. C and D, the exosomes concentration was measured by nanoparticle tracking analysis (NTA), and data were expressed as particles/ml. The data shown represent the mean ± S.D. of three independent experiments and analyzed by one way-ANOVA, ∗∗p < 0.01; E and F, ECs were treated with 100 μmol/L FAC or DFO, and after 24 h, His-FTH1 or Tf were added to the apical chamber and incubated 24 h. This experiment was designed to demonstrate that exogenously applied His-FTH1 was transcytosed via exosomes isolated from the basal chamber, and the amount of His-FTH1 found in the exosomes was altered by the iron status of the cells. The levels of Tf in the exosomes of the basal chamber were also altered by FAC and DFO treatment. E and F illustrates representative immunoblots of Tf and His-FTH1 in exosomes respectively. His-FTH1 and Tf/ponceau S expressions are presented as fold change relative to FAC (data have been normalized to the FAC treatment for each independent experiment). The data shown represent the mean ± S.D. of three independent replicates and analyzed by unpaired t test, ∗∗p < 0.01, ∗∗∗p < 0.001. 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.