Fig. 1. Effects of FGF9 on RCS cell proliferation, FGFR3 activation and gene expression. RCS cells were harvested at the indicated time points following stimulation with 20 ng/ml of human recombinant FGF9 and 1µg/ml of heparin. (A) Equal amounts of cell lysates were analyzed by SDS-PAGE and western blotting with anti-FGFR3 antibodies (upper panel), anti-pMAPK antibodies (middle panel) and anti-FRS2 antibodies (lower panel). (B) Cells were harvested, and RNA was prepared and subjected to northern blot analysis using human FGFR3 cDNA as a probe. (C) RCS cells (60,000 cells/well) were incubated with FGF9 and heparin for 0 (untreated control), 8, 16, 24 and 72 hours as indicated. Then, FGF9 was washed out and the cells were cultured further under normal conditions for 72 hours, at which point the cell number was determined. The initial cell number seeded in each system was 0.6x10⁵. The respective cell numbers obtained at the end of the 72-hour incubation were 7.2x10⁵, 6.8x10⁵, 3.5x10⁵, 2.9x10⁵ and 1.1x10⁵. Values are averages of triplicates, with standard deviation <10% in all cases.