Fig. 1

Increased levels of total and phosphorylated MET in PRCC cells. A Lysates from cell lines with gain of chromosome 7, harboring MET TK domain mutation (UOK345, H1112R; UOK337, H1106Q) or wild type MET (UOK342, UOK332) and normal RPTEC were collected and subjected to western blot analysis. Increased levels of both total and phosphorylated MET (Y1234/1235) were observed in all PRCC cell lines in comparison to RPTEC (B) Effect of selective MET TK inhibitor, EMD1214063 on viability of PRCC cells. UOK345, UOK337, UOK342, UOK332 and normal RPTEC cells were treated with a range of concentrations of EMD1214063 for 72 h in presence of 100 ng/ml HGF, and cell viability was determined using Cell-Titer Glo assay. EMD1214063 treatment had minimal effect on the proliferation of PRCC cell lines comparable to that seen in RPTEC cells. Data are expressed as mean ± SD of triplicates and are representative of three independent experiments. ns: not significant (p > 0.05), and was calculated by the two tailed Student’s t test (C) Effect of EMD1214063 on invasion in PRCC cells. Serum starved UOK345 or UOK342 cells were seeded in the upper chamber of CIM plate-16 in DMEM media containing 50 or 500 nM of EMD1214063 or vehicle. In the bottom chamber, DMEM with 100 ng/ml HGF or without HGF (negative control) were added and the inhibition of invasion relative to vehicle alone–treated cells was determined. The impedance value of each well was continually monitored every 15 min by the xCELLigence system for 24 h recorded as cell index. EMD1214063 treatment at either dose had no effect on the invasion of PRCC cell lines.. ns: not significant (p > 0.05), and was calculated by the two tailed Student’s t test. D Effect of EMD1214063 on phosphorylation of MET and downstream signaling molecules. EMD1214063 inhibits pMET(Y1234/1235) but no significant effect was observed on downstream signaling molecules pAKT (Ser473) and pERK (Thr202/Tyr204) in PRCC cell lines. UOK345 and UOK342 cells were treated with EMD1214063 (50 nM) for 2 h followed by the addition of HGF (100 ng/ml) for 15 min, lysed and subjected to western blot analysis. Three independent experiments were performed, and a representative result is shown. *p < 0.05 is considered significant and was calculated by the two tailed Student’s t test or other tests