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Files in this Data Supplement:
Fig. S1. Trans-endocytosis of SHPS1 on SHPS1-expressing cells by neighboring CD47-expressing cells and trans-endocytosis of full-length CD47 with SHPS1. (A-F) CHO-SHPS1 and CHO-CD47 cells were cocultured for 1 hour, fixed, and subjected to immunostaining with a mAb to SHPS1 (green) in the presence (A) or absence (D) of 0.1% Triton X-100. The cells were then stained with Rhodamine-conjugated phalloidin (red; B,E) in the presence of 0.1% Triton X-100. Merged images are shown in C and F. Arrows indicate SHPS1-positive vesicles in CD47-expressing cells. Dotted lines in A and D indicate CHO-CD47 cells. Bar, 20 µm. (G) CHO-SHPS1 and CHO-CD47 cells were cocultured, fixed, and stained as in A-F. The percentage of cells with SHPS1-positive vesicles was determined for CHO-CD47 cells adjacent to CHO-SHPS1 cells by assay A (see Materials and Methods). (H) CHO-SHPS1 and CHO-CD47 cells were cocultured as in A-F, fixed, and stained with a mAb to SHPS1 before and after permeabilization with 0.1% Triton X-100 (see Materials and Methods). The numbers of surface, internalized and total SHPS1-positive vesicles in CHO-CD47 cells adjacent to SHPS1-expressing cells were determined by assay B (see Materials and Methods). Data in G and H are means ± s.e.m. from three separate experiments. *P<0.05. (I-N) CHO-Ras cells were transiently transfected with an expression vector for CD47 tagged at its C-terminus with the Myc epitope. Twenty-four hours after transfection, the cells were cultured with CHO-SHPS1 cells for 1 hour. All the cells were then fixed and subjected to two-color immunostaining with the mAb miap301 to the extracellular region of CD47 (green; I) and with the mAb 9E10 to the Myc epitope (red; J) in the presence of 0.1% Triton X-100. Alternatively, the cocultured cells were double-stained with a mAb to SHPS1 (green; L) and with that to Myc (red; M) in the presence of 0.1% Triton X-100. Merged images are shown in K and N. Arrows indicate CD47-positive vesicles in SHPS1-expressing cells (I-K) or vesicles positive for both SHPS1 and CD47 in SHPS1-expressing cells (L-N). The dotted lines in I, J, K and M, and in L, indicate CHO-SHPS1 cells and a CD47-Myc-expressing cell, respectively. Bar, 20 µm. The observation that CD47-positive vesicle-like structures in CHO-SHPS1 cells that formed contacts with the CHO-Ras cells expressing CD47-Myc were also stained with 9E10 suggests that the full-length CD47 molecule undergoes trans-endocytosis by neighboring SHPS1-expressing cells (I-K). In addition, the finding that most CD47-Myc-positive vesicle-like structures in CHO-SHPS1 cells were also recognized by the mAb to SHPS1 (L-N) indicates that SHPS1 was endocytosed together with bound CD47.
Fig. S2. Relative expression levels of SHPS1 and CD47 in CHO-Ras cell lines. (A,B) COS-7 cells were transfected with an expression vector for SHPS1 tagged at its C-terminus with the Myc epitope (SHPS1-Myc; A) or that for CD47 tagged with the Myc epitope (CD47-Myc; (B). Twenty-four hours after transfection, cells were lysed and resulting lysates (3-12 µg of protein) were subjected to immunoblot analysis with the 9E10 mAb to Myc and either p84 mAb to SHPS1 (A, left) or the miap301 mAb to CD47 (B, left) by the use of the ECL detection system. The intensity of each band detected by p84, miap301 or 9E10 was determined by densitometric analysis. The ratios of the intensity of either a p84-reactive band (A) or miap301-reactive band (B) to the intensity of corresponding 9E10-reactive band were then calculated from four different conditions (3-12 µg of total protein in each lane) and the collected values were then averaged. Data (A,B, right) are means ± s.e.m. from a representative experiment (9E10:p84:miap301 was 1:0.13±0.02:0.15±0.04). (C) Lysates (3-12 µg of protein) of CHO-SHPS1 or CHO-CD47 cells were subjected to immunoblot analysis with either the p84 mAb or the miap301 mAb (left panels) as described above. The intensity of each band detected by p84 or miap301 was determined by densitometric analysis. Each value obtained with either p84 or miap301 was then divided by 0.13 for the p84 bands or 0.15 for the miap301 bands. The ratios of such corrected values for miap301 to the corresponding values for p84 were then calculated from four different conditions (3-12 µg of total protein) and they were averaged. Data are means ± s.e.m. from a representative experiment.
Fig. S3. Cell-surface expression of SHPS1 and its mutants. CHO-Ras cell lines expressing wild-type or mutant forms of SHPS1 were subjected to immunofluorescence analysis with the p84 mAb to SHPS1 under nonpermeabilized condition, and the immunoreactivity of SHPS1 was quantified. The averaged signal intensity, of fluorescence per pixel, was measured using the software of the LSM5 Pascal microscope. The averaged background intensity, which was calculated from the immunofluorescence signals of parental CHO-Ras cells, was subtracted from the values obtained for each cell line. Data are means ± s.e.m. from 50 different cells. *P<0.05. a.u., arbitrary units.
Fig. S4. Effect of expression level of SHPS1 on the number of trans-endocytosed vesicles containing CD47. (A) Lysates (10 µg of protein) of two different CHO-SHPS1 cell lines expressing SHPS1 at a high or low level were subjected to immunoblot analysis with the p84 mAb to SHPS1. (B) The two CHO-SHPS1 cell lines were separately cocultured with CHO-CD47 cells, fixed and stained as in Fig. 1J-L. The numbers of surface, internalized and total CD47-positive vesicles in CHO-SHPS1 cells adjacent to CHO-CD47 cells were determined by assay B. Data are means ± s.e.m. from a representative experiment. *P<0.05 versus corresponding value for cells with the high level of SHPS1 expression. (C) The percentages of the total number of CD47-positive vesicles (Ntotal) that were present at the cell surface (Nsur) or inside the cell (Nin) were determined from the mean values in B. An increase in the expression level of SHPS1 thus increased the number of trans-endocytosed vesicles containing CD47 (B), but it did not alter the percentage of such vesicles present either at the cell surface or intracellularly (C).
Fig. S5. Effect of forced expression of a dominant negative mutant of Rac or NWASP-CRIB on cell-surface expression of SHPS1. CHO-Ras cells were cotransfected with an expression vector for SHPS1 and with a vector either for Rac(T17N) or for NWASP-CRIB (or with the corresponding empty vector). Twenty-four hours after transfection, the cells were fixed and subjected to immunofluorescence analysis with the p84 mAb to SHPS1 under nonpermeabilized condition and SHPS1 immunoreactivity was quantified as in Fig. S3. Data are means ± s.e.m. from 50 different cells. *P<0.05. a.u., arbitrary units.
Fig. S6. Downregulation of SHPS1 and CD47 in cocultures of CHO-SHPS1 and CHO-CD47 cells. CHO-SHPS1 and CHO-CD47 cells were mixed and cocultured for 2 hour. In control experiments, CHO-SHPS1 cells (left panels) or CHO-CD47 cells (right panels) were cocultured with parental CHO-Ras cells. Total lysates (22 µg of protein) of cocultured cells were subjected to immunoblot analysis with the p84 mAb to SHPS1 (upper left panel) or the miap301 mAb to CD47 (upper right panel). The lysates were also subjected to immunoblot analysis with a mAb to β-tubulin (lower panels).
Fig. S7. Accumulation of CD47 at sites of contact between neurites and HEK-SHPS1 cells expressing a dominant negative mutant of dynamin. (A-C) Mouse hippocampal neurons were cocultured for 1 hour with HEK-SHPS1 cells transfected with an expression vector for the dominant negative mutant dynamin-1(K44A). The cells were then fixed and stained in the presence of 0.1% Triton X-100 with mAbs to CD47 (red; A) and to the HA epitope tag of dynamin 1(K44A) (green; B). A merged image is shown in C. As a control, neurons were cocultured with HEK-SHPS1 cells transfected with an expression vector for GFP, which were subsequently identified by GFP fluorescence (data not shown). Arrows indicate accumulation of CD47 immunoreactivity at the sites of contact between HEK-SHPS1 cells and neurites. Bar, 20 µm. (D) CD47 immunoreactivity at the contact sites of cells treated as in A-C was quantified by tracing a 1-pixel-wide linear segment along a neurite (∼100 µm) from the distal end, where there was no contact with HEK-SHPS1 cells, to such a contact site. The average fluorescence signal intensity per micrometer of each segment was measured using the software of the LSM5 Pascal microscope. The average background intensity (based on neighboring regions that did not contain any neurites or HEK-SHPS1 cells) was subtracted from the values obtained for neurites. The ratio of the average fluorescence intensity for contact sites versus noncontact sites of the same neurite was calculated for 50 different neurites and averaged. Data are means ± s.e.m. *P<0.05.
Movie 1. Time-lapse movie of the data shown in Fig. 6A-J. Images were captured every 1 minute. The frame rate of the movie is ten frames per second.
Movie 2. Time-lapse movie of the data shown in Fig. 6K-N. Images were captured every 1 min. The frame rate of the movie is ten frames per second.
Movie 3. Trans-endocytosis of CD47 on primary cultured hippocampal neurons by neighboring glia-like cells. Hippocampal neurons (and glial cells) were transfected with an expression vector for CD47-YFP as in Fig. 8M-N. Twelve hours after transfection, fluorescence images of CD47-YFP (Green) and light-field images were acquired every 1 minute and merged. CD47-YFP−positive vesicles were generated at sites of cell-cell contact between transfected neurons and neighboring glia-like non-neural cells (indicated by an arrow). The frame rate of the movie is ten frames per second.
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