Iron repletion leads to the colocalization of CD63 and NCOA4 into the same vesicle. (A) IMR90SV cells were incubated in medium (–FBS) (control) or medium supplemented with FAC (10 µg/mL) for 16 hours at 37°C. Cells were fixed and stained with anti-NCOA4 (green) and anti-CD63 (red) antibodies. (B) Quantification of CD63 and NCOA4 intensity in panel A. (C) Quantification of CD63 and NCOA4 colocalization indicated by Manders’ overlap coefficient. (D) Control and NCOA4KD IMR90SV cells were incubated for 16 hours at 37°C with 10 µg/mL FAC. Cells were fixed and stained with anti-FTH (green) and anti-CD63 (red) antibodies. (E) Quantification of CD63 and NCOA4 intensity in panel D. (F) Quantification of CD63 and FTH colocalization by Manders’ overlap coefficient. (A,D) Thin scale bar, 10 µm; thick scale bar, 2.5 µm. For analysis, >60 cells were examined per condition over 3 experiments. Results are mean ± SEM (3 experiments). Significance was determined using the Student t test. **P < .01; ****P < .0001.