Fig. 2.

A: Activation of FGFR3 signaling cascade in wild-type (wt) FGFR3 harboring cell lines UM-UC1 by stimulation with fibroblast growth factor (FGF)-1. Cells were cultured in regular growth medium on 6 cm culture dishes to about 50% confluence. Cells were starved in serum free media for 24 hours. Growth medium was exchanged to medium supplemented with FGF-1 at a concentration of 10ng/ml. Cells were then harvested on ice according to the time point of 2, 5, 10, 15 and 20 minutes after stimulation. Cells were lysed with RIPA buffer including proteinase and phosphatase inhibitor and 60 ug of total protein used. Indicating for an activation of the FGFR3 signaling cascade is shown by a time dependent increase of phospho-FGFR, followed by phosphorylation of FRS-2, ERK-1/2 and Akt.

Inhibition of FGFR3 signaling by the FGFR3 specific inhibitory antibody R3Mab in wt-FGFR3 harboring UM-UC1 cells and the S249C mutant FGFR3 bearing cell lines UM-UC14. B: UM-UC1 cells were plated in normal growth media and allowed to attach for 24 hours, then washed two times with PBS and then starved in media without FBS for 24 hours, followed by stimulation with FGF-1 (15ng/ml) for 10 min in the presence of Heparin (10 ug/ml). Either human control IgG or R3Mab was added in serum free media at concentrations of 10 and 50 ug/ml. Phosphorylation of FGFR, FRS2, Erk1//2 and Akt was evaluated by Western blot and demonstrates lower phosphorylation levels under treatment with R3Mab. C: A strong auto-phosphorylation of FGFR3 is found in UM-UC14 cells in regular growth conditions. R3Mab blocks auto-phosphorylation of FGFR3^S249C receptor even under stimulated conditions with supplemented FGF1, while Erk1/2 shows increased levels of phosphorylation under stimulation with FGF1 and at low concentrations of R3Mab (1 ug/ml). D: In vitro growth inhibition of R3Mab on urothelial cancer cell lines. Cell lines were grown under to 50% confluence in regular growth medium. R3Mab was then added to growth medium and growth inhibition was evaluated by crystal violet staining after 48 hours of treatment with FGFR3 specific inhibitory antibody. Mesenchymal cell lines with low expression of FGFR3 show no specific response to treatment with R3Mab at concentrations up to 100 ug/ml, while epithelial cell lines with high expression of FGFR3 show a growth inhibition of up to 50%, as observed in UM-UC1 cells, a cell line derived from a lymph node metastasis from an urothelial carcinoma.