Figure 2.

Overexpression of secreted protein acidic and rich in cysteine (SPARC) in Daoy cells inhibits tumour-induced angiogenesis in vitro and in vivo. (A) In vitro angiogenesis: Daoy-P, Daoy-EV and Daoy-SP cells (2 × 10⁴ per well), either with SPARC siRNA treatment or with anti-SPARC antibody treatment, were seeded in eight-well chamber slides. After 24 h, the medium was removed and 4 × 10⁴ HMEC cells were added. The cells were allowed to co-culture for 36 h the cells were fixed and performed immunofluorescence for factor-VIII as described in the ‘Materials methods’ section and observed for angiogenic response. Relative branch points and relative tube length were quantified as described in the ‘Materials and methods’ section. Columns, mean of triplicate experiments; bars, s.e.; ^*P<0.01, significant difference from Daoy-P cells; ^**P<0.01, significant difference from Daoy-EV cells treated with SPARC siRNA or anti-SPARC antibody (siRNA=SPARC siRNA; anti-SP=anti-SPARC antibody). (B) In vivo angiogenesis: Daoy-P, Daoy-EV and Daoy-SP cells (1 × 10⁶) were implanted into diffusion chambers and were surgically placed underneath the dorsal skin of athymic nude mice as described in the ‘Materials and Methods’ section. PV, pre-existing vasculature; TN, tumour-induced vasculature. (C) Newly formed vessels were quantified and represented as per field. Columns, mean of triplicate experiments; bars, s.e.; ^*P<0.01, significant difference from Daoy-EV cells. (D) Western blot analysis showing SPARC and vascular endothelial growth factor (VEGF) levels with the treatment of siRNA against SPARC. GAPDH served as loading control.