Figure 5. Inhibition of COL1A1/PDGFB fusion oncogene-mediated PDGFR activation and NIH3T3 cell transformation.

(A) Parental NIH3T3 and NIH3T3^COL1A1/PDGFB cells were treated with the indicated concentrations of roneparstat (Rone) and the drug antiproliferative activity was assessed 72 h later by cell counting. The right panel shows the effect of 24 h drug treatment on the transformed morphologic phenotype of NIH3T3^COL1A1/PDGFB cells in comparison with parental cells. Representative images were taken under a phase-contrast microscope (original magnification, 100X). (B) Inhibition of anchorage-independent growth of NIH3T3^COL1A1/PDGFB cells. Cells were seeded in soft agar in the presence or absence of increasing roneparstat concentrations. Colonies were counted after 11 days using a magnifying projector and data reported as mean colony number/field ± SD. (C) Inhibition of NIH3T3^COL1A1/PDGFB cell invasive ability. After 24 h of exposure to roneparstat (1 mg/ml), transfected fibroblasts were transferred to Matrigel-coated transwell chambers in serum-free medium and invasion assessed 24 h later. Data are reported as the average cell number per field ± SD. Representative images of SRB-stained invaded cells are shown beside (original magnification 100X). (D) Effect of roneparstat (1 mg/ml, 24 h) on PDGFR activation and signaling. The receptor was immunoprecipitated from NIH3T3^COL1A/PDGFB cell lysates with an anti-PDGFRβ antibody and its activation assessed by western blotting using an antibody recognizing tyrosine phosphorylated PDGFR. In the same filter, anti-phosphotyrosine antibody (pY) revealed phosphopeptides co-immunoprecipitated with PDGFRβ, among which FAK, which was then identified by blotting with anti-phospho-FAK and anti-FAK antibodies. Blots performed on cell lysates (CL) show the protein overall levels and loading control. (E) Indirect immunofluorescence showing, on the left, localization of tyrosine phosphorylated PDGFR in control and roneparstat-treated (1 mg/ml for 24 h) NIH3T3^COL1A/PDGFB cells. On the right, cellular distribution of F-actin stained with green fluorescent phalloidin. Nuclei are evidenced with Hoechst 3341 counterstaining (blue). Two images for each sample are shown. Original magnification, 1000X. **P < 0.01, ***P < 0.001 drug-treated versus untreated control cells. Data from representative experiments, performed in duplicate, are shown.