Figure 2.
CD63 expression is increased by cellular iron loading and decreased by iron depletion. (A) IMR90SV cells were incubated for 16 hours at 37°C with either control medium or medium containing FAC (5 or 20 µg/mL) without FBS (–FBS). Cells were then lysed in lysis buffer, and 20 µg of protein from each sample was analyzed with anti-CD63, anti-CD81, anti-FTH, anti-Lamp2, and anti–β-actin antibodies using immunoblotting. (B) Quantification of CD63, CD81, and Lamp2 intensity from panel A. (C) IMR90SV cells were incubated with 10 µg/mL FAC or 50 µM DFX for 6 hours at 37°C in media (–FBS). The relative mRNA levels of CD63, CD81, FTH, and FTL were calculated using the 2-ΔΔCq method and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (D) IMR90SV cells were incubated with 10 µg/mL FAC for 6 hours at 37°C in media (–FBS). Cells were then lysed in lysis buffer, and 20 µg of each sample was analyzed with anti-CD63, anti-CD81, anti-FTH, anti-FTL, and anti–β-actin antibodies using immunoblotting. (E) IMR90SV cells were incubated with either control medium (–FBS) or medium containing 10 µg/mL FAC or 5 µM DFX for 16 hours at 37°C. Cells were fixed and stained with anti-CD63 antibodies (red) and 4′,6-diamidino-2-phenylindole (DAPI; blue). As a negative control, IMR90SV cells were incubated with 10 µg/mL FAC for 16 hours at 37°C and were treated with secondary antibody without primary antibody. Scale bar, 10 µm. (F) Quantification of CD63 intensity per cell was performed with >60 cells examined per condition from 3 experiments. (G) IMR90SV cells were incubated with medium (–FBS) (control), with 10 µg/mL FAC (–FBS), 10% FBS complete medium (complete), or complete medium containing 2 nM bafilomycin A1 (Baf A1/complete) for 12 hours at 37°C. Then, 20 µg of protein per sample was examined using immunoblotting analysis. (H) Quantification of CD63, CD81, FTH, and LC3-II intensity of entities in panel G. (I) Confocal immunofluorescence microscopy of IMR90SV cells. Cells were cultured with medium (–FBS), medium containing 2 nM Baf A1 (–FBS), or medium containing 10 µg/mL FAC (–FBS) for 12 hours at 37°C. Cells were then fixed and stained with anti-CD81 (green), anti-CD63 (red), and DAPI (blue). Thin scale bar, 10 µm; thick scale bar (for enlarged photos), 2.5 µm. (J) Quantification of CD63 or CD81 intensity per cell. (K) Calculation of the Manders’ overlap coefficient assessing CD63 and CD81 overlap. For panels J and K, data are from examining >60 cells per condition over 3 experiments. Quantification of CD81 and CD63 colocalization was determined via the Manders’ overlap coefficient. All results are mean ± standard error of the mean (SEM) from 3 representative experiments. Significance was determined using the Student t test. *P < .05; **P < .01; ***P < .001; ****P < .0001. n.s., not significant.

CD63 expression is increased by cellular iron loading and decreased by iron depletion. (A) IMR90SV cells were incubated for 16 hours at 37°C with either control medium or medium containing FAC (5 or 20 µg/mL) without FBS (–FBS). Cells were then lysed in lysis buffer, and 20 µg of protein from each sample was analyzed with anti-CD63, anti-CD81, anti-FTH, anti-Lamp2, and anti–β-actin antibodies using immunoblotting. (B) Quantification of CD63, CD81, and Lamp2 intensity from panel A. (C) IMR90SV cells were incubated with 10 µg/mL FAC or 50 µM DFX for 6 hours at 37°C in media (–FBS). The relative mRNA levels of CD63, CD81, FTH, and FTL were calculated using the 2-ΔΔCq method and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (D) IMR90SV cells were incubated with 10 µg/mL FAC for 6 hours at 37°C in media (–FBS). Cells were then lysed in lysis buffer, and 20 µg of each sample was analyzed with anti-CD63, anti-CD81, anti-FTH, anti-FTL, and anti–β-actin antibodies using immunoblotting. (E) IMR90SV cells were incubated with either control medium (–FBS) or medium containing 10 µg/mL FAC or 5 µM DFX for 16 hours at 37°C. Cells were fixed and stained with anti-CD63 antibodies (red) and 4′,6-diamidino-2-phenylindole (DAPI; blue). As a negative control, IMR90SV cells were incubated with 10 µg/mL FAC for 16 hours at 37°C and were treated with secondary antibody without primary antibody. Scale bar, 10 µm. (F) Quantification of CD63 intensity per cell was performed with >60 cells examined per condition from 3 experiments. (G) IMR90SV cells were incubated with medium (–FBS) (control), with 10 µg/mL FAC (–FBS), 10% FBS complete medium (complete), or complete medium containing 2 nM bafilomycin A1 (Baf A1/complete) for 12 hours at 37°C. Then, 20 µg of protein per sample was examined using immunoblotting analysis. (H) Quantification of CD63, CD81, FTH, and LC3-II intensity of entities in panel G. (I) Confocal immunofluorescence microscopy of IMR90SV cells. Cells were cultured with medium (–FBS), medium containing 2 nM Baf A1 (–FBS), or medium containing 10 µg/mL FAC (–FBS) for 12 hours at 37°C. Cells were then fixed and stained with anti-CD81 (green), anti-CD63 (red), and DAPI (blue). Thin scale bar, 10 µm; thick scale bar (for enlarged photos), 2.5 µm. (J) Quantification of CD63 or CD81 intensity per cell. (K) Calculation of the Manders’ overlap coefficient assessing CD63 and CD81 overlap. For panels J and K, data are from examining >60 cells per condition over 3 experiments. Quantification of CD81 and CD63 colocalization was determined via the Manders’ overlap coefficient. All results are mean ± standard error of the mean (SEM) from 3 representative experiments. Significance was determined using the Student t test. *P < .05; **P < .01; ***P < .001; ****P < .0001. n.s., not significant.

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