Figure 3

Inhibition of endogenous SPARC and its effect on cell migration, colony-formation, xenograft growth, and FGF-induced proliferation. (A) Migration assay. The number of cells that moved to the lower side of the Boyden chamber was determined as described in the legend of Figure 1 for wild-type ASPC-1 and PANC-1 cells, vector alone (control)-transfected clones (AN-9, PN-21), and SPARC shRNA-expressing clones (A2-7, A3-3, P1-15, P1-20), respectively. The results are expressed as mean number (±s.d.) of migrated cells within 36 h of three separate experiments. ^*P<0.05 compared with wild-type and control-transfected cells. (B) Soft-agar assay. Colony-formation of indicated cells was determined after 14 days by staining and counting viable colonies. The results are mean number of colonies per dish (±s.e.) of triplicate determinations from five separate experiments. ^*P<0.05 compared with wild-type and control-transfected cells. (C) Xenograft formation. Indicated PANC-1 cells (10⁶ per site) were subcutaneously injected into nude mice. Tumour growth was followed for 80 days. Eleven sites of 16 injected sites of PN-21 and 6 of 8 of PN-22 control-transfected clones and 12 of 16 of P1-20 and 8 of 8 of P1-15 SPARC shRNA-expressing clones developed tumours. Shown is the mean tumour volume for developed nodules: PN-21 (n=11), PN-22 (n=6), P1-15 (n=8), P1-20 (n=12). ^*P<0.05 compared with PN-21. (D) FGF-induced proliferation. Wild-type PANC-1, control-transfected (PN-21), and SPARC shRNA (P1-15, P1-20)-expressing cells were cultured for 48 h in serum-free medium in the presence of heparin alone (1 μg ml^–1), 5 nM FGF1 or FGF2 in the presence of heparin, or 5 nM EGF. Proliferation was determined by the MTT assay. The results are shown as mean growth (in %) of respective untreated control and are means (±s.e.) of quadruplicate determinations from three separate experiments. ^*P<0.05 compared with the effects of ligand in wild-type and control-transfected cells.