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Fig. 1. Trans-endocytosis of CD47 on CD47-expressing cells by neighboring SHPS-1-expressing cells. (A-I) CHO–SHPS-1 or CHO-Ras (CHO) cells were cocultured with CHO-CD47 cells for 1 hour. The cells were then fixed and subjected to immunostaining with a mAb to CD47 (green; A,D,G) and staining with Rhodamine-conjugated phalloidin (red; B,E,H) in the presence of 0.1% Triton X-100. Merged images are shown in C, F and I. Arrows indicate CD47-positive vesicle-like structures in SHPS-1-expressing cells, and the dotted lines in A, D and G indicate CHO–SHPS-1 (A,D) or CHO-Ras (G) cells. Bar, 20 µm. (J-L) CHO–SHPS-1 and CHO-CD47 cells were cocultured as in A-I, fixed, and stained sequentially with a mAb to CD47 before (green; J) or after (red; K) permeabilization with 0.1% Triton X-100 (see Materials and Methods). The merged image is shown in L. The dotted lines in J-L indicate a CHO–SHPS-1 cell. Bar, 20 µm. (M) CHO–SHPS-1 and CHO-CD47 cells were cocultured for 1 hour, fixed, and stained with a mAb to CD47 in the absence or presence of 0.1% Triton X-100. The percentage of cells with CD47-positive vesicles was determined for CHO–SHPS-1 cells adjacent to CD47-expressing cells by assay A (see Materials and Methods). The effect of treatment of CHO-CD47 cells with the miap301 mAb to CD47 (50 µg/ml) for 15 minutes before and during coculture was also determined. (N) CHO–SHPS-1 and CHO-CD47 cells were cocultured, fixed, and stained as in J-L. The numbers of surface, internalized, and total CD47-positive vesicles in CHO–SHPS-1 cells adjacent to CHO-CD47 cells were determined by assay B (see Materials and Methods). Data in M and N are means ± s.e.m. from three separate experiments. **P<0.01.
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