Figure 3

Effect of Zt/f2 on RON/RON160 phosphorylation, cell proliferation, and migration by HT-29 cells. A) Serum-starved HT-29 cells (3 × 10⁶ cells/sample) were stimulated for 15 min at 37°C by 2 nM MSP, 10 μg/ml Zt/g4, or both followed by immunoprecipitation using Zt/g4. Cells treated with control IgG served as the control. Phospho-RON/RON160 was determined by Western blot analysis using anti-PY100 mAb. Phosphorylation of Erk1/2 and AKT were determined by Western blot analysis using specific antibodies. B) HT-29 cells in wells of a 96-well plate (10,000 cells/well) were treated with MSP (2 nM), Zt/f2 (10 μg/ml), or both in triplicate for three days. Cells numbers were counted as detailed in Materials and Methods. C) A 48-well cell migration chamber was used to determine levels of HT-29 cell migration in a period of 24 h. The bottom wells of the chamber were filled with MSP (2 nM), Zt/f2 (10 μg/ml), or both. The top wells were filled with HT-29 cells (1 × 10⁴ cells/well) in DMEM with 5% FBS. Cells migrated and attached to the membrane were counted as previously described (Wang et al., 1994).