Yeh et al. BMC Cancer 2011, 11:139 http://www.biomedcentral.com/1471-2407/11/139 A Page 5 of 12 Figure 2 Expression of c-Met, phosphorylated c-Met, Axl and PDGFR-a in NIH/3T3 and NIH-Met5 cells in the presence or absence of tetracycline. A: A 192 RTK gene cDNA chip was used to screen the gene expression profiles which are positively correlated with c-Met in NIH/ 3T3 and inducible NIH-Met5 cell lines. A total of 8 RTK genes were shown to positively correlate with c-Met over-expression by CAST analysis (Methods). Only the gene expression profiles of c-Met, Axl and PDGFR-a are shown. NIH/3T3 (1D): the parental NIH/3T3 cell without Tet for 24 h; NIH-Met5 (1D): NIH-Met5 cell without Tet for 24 h; 4D: NIH-Met5 cell with Tet for 24 h followed by Tet-free treatment for another 4 days; 7D: NIH-Met5 cell with Tet for 24 h followed by Tet-free treatment for another 7 days. Relative expression level of each gene is obtained as compared with the common reference RNA [47] B: The expression levels of c-Met, p-Met, Axl and PDGFR-a were evaluated in NIH/3T3 and NIH- Met5 cells without Tet treatment (lanes 1 and 3), or after treatment for 24 h first and then replaced with Tet-free medium for additional 4 and 7 days, (lanes 4 and 5) by Western blotting. b-actin was used as an internal control. The numbers under each band represent the relative intensity. The regulation was then examined at protein level in NIH-Met5 cells. As shown in figure 2B (lane 3), c-Met was overexpressed in the absence of Tet, while sup- pressed c-Met expression was demonstrated after treat- ment of Tet for 24 h, as with that of figure 1. A reversion of c-Met expression gradually appeared after removal of Tet for 4 and 7 days. Expression of c-Met became visible by day 4 and almost completely reversed by day 7 after removal of Tet (Figure 2B, lanes 4 and 5). The parental NIH/3T3 cells were used as a control (Fig- ure 2B, lanes 1 and 2). Using total c-Met and p-Met as the reference, expres- sion of Axl and PDGFR-a showed a comparable trend to that of c-Met at day 4 and day 7 (Figure 2B, lanes 4 - 5), respectively. This positive association of Axl or PDGFR-a with c-Met expression was also demonstrated in T24-Met3 human bladder cancer cell line (Figure 1B). However, no difference of Axl and PDGFR-a expression was detected in NIH3T3 cells (Figure 2B, lanes 1 and 2). Taken together, expression patterns of total c-Met and p-Met were positively correlated with Axl and PDGFR-a expression, suggesting a functional relationship between Axl/PDGFR-a and c-Met. Correlation of c-Met expression with Axl and PDGFR-a status in human bladder cancer cells Both UB40 and UB47 are two bladder cancer cell lines established locally from primary bladder cancer of superficial and muscle-invasive type, respectively [6]. Apparent expression of c-Met and p-Met protein was detected in these two cell lines, and both Axl and PDGFR-a also showed a comparable expression pat- tern (Figure 3A). To confirm their functional interac- tion, these cell lines were maintained under serum starvation for 12 h, and then treated with HGF (30 ng/ml) for 10 min (Figure 3A). Up-regulation of Axl and PDGFR-a was demonstrated in UB40 and UB47 cells after HGF stimulation with a corresponding increase of p-Met (Figure 3A). Level of p-Met posi- tively correlated with the expression of Axl and PDGFR-a, suggesting a relationship among c-Met, Axl and PDGFR-a.