Figure 6.

TSP1 inhibits proliferation of HUVECs. A-B, β_1C-PC3 cells were cultured either in the absence (-tet, left panel) or presence (+tet, right panel) of tet. Cells were incubated in the presence or absence of TSP1 (0, 0.1, 1 or 10 μg/ml). After 24 h of incubation, cells were processed to measure cell proliferation using Sulforhodamine B (SRB) assay (A). Cells were detached, plated on FN and attached cells were incubated in the presence of CHX, TNF-α or TSP1 (2 μg/ml). Cells were detached and processed for DNA fragmentation assay (B). C, HUVECs were plated on 96-well plate and incubated overnight for attachment. Cells were washed with PBS and incubated in serum free medium for 6 h. HUVECs were treated with conditioned media from β_1C-PC3 (clone C1, -tet or +tet) or β_1A-PC3 (clone A2, -tet or +tet) transfectants for 24 h. Cell proliferation was measured using SRB. TSP1 secreted by β_1C-PC3 cells inhibits proliferation of HUVECs (*p<0.05) (left panel). C, right panel, cells were plated and treated with conditioned media from β_1C-PC3 (-tet) or β_1A-PC3 (-tet) as described above in the presence of either mIgM or TSP Ab (A4.1, 20 μg/ml) and proliferation was measured using SRB after 48 h. The TSP1 inhibitory Ab prevents the effect of β_1C-PC3 conditioned medium on proliferation of HUVECs (*p<0.05). Grey bars, conditioned medium; black bars, HUVEC culture medium. D, β_1C-PC3 transfectants (clones C1 and C2) were cultured in the presence or absence of tet. Cells were processed to measure anchorage-independent growth in the presence of either mIgM or a-TSP (TSP blocking Ab, clone A4.1). The expression of β_1C suppresses anchorage-independent growth of PC3 cells (*p<0.05).