Figure 3.
Increasing cellular iron after incubation with FAC upregulates CD63, FTH, and FTL expression in isolated EVs with the ferritin in EVs containing iron. (A) IMR90SV cells were incubated with medium containing 0 (control), 5, or 20 µg/mL FAC for 48 hours at 37°C. (A) EVs and (C) EVs or cells were prepared in lysis buffer, the protein concentration was measured by a bicinchoninic acid (BCA) protein assay, and 20 µg of protein from each sample was analyzed using anti-CD63, anti-CD81, anti-FTH, anti-FTL, anti-calnexin, and anti-protein disulphide-isomerase (PDI) antibodies. (B) Quantification of CD63, CD81, FTH and FTL shown in panel A using Fiji software. Expression was normalized by Ponceau S staining as in panel A. (D) Representative transmission electron microscope images of isolated EVs from IMR90SV cells with negative staining by uranyl acetate. Red arrowheads show EVs. Scale bar, 100 nm. (E) Quantification of EV number per field in panel D. Thirty transmission electron micrograph fields over 3 experiments (>90 fields) at an original magnification of 20 000× were randomly chosen, and EVs were counted by a blinded investigator (up to 500 to 1000 vesicles were counted per experiment). (F) IMR90SV cells incubated with FAC (10 µg/mL) for 48 hours at 37°C express high levels of FTH and FTL in cells and EVs, as demonstrated by native polyacrylamide gel electrophoresis (PAGE) and immunoblotting (20 µg protein per well; western blotting (WB), left-hand membranes). The same samples were simultaneously run under native PAGE conditions and stained with Prussian blue (60 µg of protein/lane) to identify Fe+3. Coomassie blue staining of the gel was used to verify equal protein loading; full-lane densitometry of staining showed no difference with or without FAC treatment. (G) Ratios of FTH and FTL expression in EVs and cells after densitometric analysis of the results in panel F. (H) Iron:FTH or iron:FTL ratios in cells or EVs after densitometric analysis of the results in panel F. Results are mean ± SEM (3 experiments). Significance was determined using the Student t test. *P < .05; **P < .01; ***P < .001; ****P < .0001.

Increasing cellular iron after incubation with FAC upregulates CD63, FTH, and FTL expression in isolated EVs with the ferritin in EVs containing iron. (A) IMR90SV cells were incubated with medium containing 0 (control), 5, or 20 µg/mL FAC for 48 hours at 37°C. (A) EVs and (C) EVs or cells were prepared in lysis buffer, the protein concentration was measured by a bicinchoninic acid (BCA) protein assay, and 20 µg of protein from each sample was analyzed using anti-CD63, anti-CD81, anti-FTH, anti-FTL, anti-calnexin, and anti-protein disulphide-isomerase (PDI) antibodies. (B) Quantification of CD63, CD81, FTH and FTL shown in panel A using Fiji software. Expression was normalized by Ponceau S staining as in panel A. (D) Representative transmission electron microscope images of isolated EVs from IMR90SV cells with negative staining by uranyl acetate. Red arrowheads show EVs. Scale bar, 100 nm. (E) Quantification of EV number per field in panel D. Thirty transmission electron micrograph fields over 3 experiments (>90 fields) at an original magnification of 20 000× were randomly chosen, and EVs were counted by a blinded investigator (up to 500 to 1000 vesicles were counted per experiment). (F) IMR90SV cells incubated with FAC (10 µg/mL) for 48 hours at 37°C express high levels of FTH and FTL in cells and EVs, as demonstrated by native polyacrylamide gel electrophoresis (PAGE) and immunoblotting (20 µg protein per well; western blotting (WB), left-hand membranes). The same samples were simultaneously run under native PAGE conditions and stained with Prussian blue (60 µg of protein/lane) to identify Fe+3. Coomassie blue staining of the gel was used to verify equal protein loading; full-lane densitometry of staining showed no difference with or without FAC treatment. (G) Ratios of FTH and FTL expression in EVs and cells after densitometric analysis of the results in panel F. (H) Iron:FTH or iron:FTL ratios in cells or EVs after densitometric analysis of the results in panel F. Results are mean ± SEM (3 experiments). Significance was determined using the Student t test. *P < .05; **P < .01; ***P < .001; ****P < .0001.

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