Yeh et al. BMC Cancer 2011, 11:139 http://www.biomedcentral.com/1471-2407/11/139 (Tet) (Figure 1A, lane 3 and 4; Figure 1B, lane 1). When c-Met was over-expressed, the increase of its phosphory- lated form (p-Met) indicates an auto-phosphorylation (Figure 1A, lane 3; Additional file 1, lane 6). Expression of p-Met was further enhanced 10 min after treatment with hepatocyte growth factor (HGF; Sigma, St. Louis, MO, USA) (Figure 1A, lane 4; Additional file 1, lane 7). In contrast, parental NIH/3T3 cells did not express c- Met and p-Met (Figure 1A, lanes 1 and 2; Additional file 1, lane4 1-4). It is interesting to note that c-Met or p-Met was not expressed in NIH-Met5 cells when trea- ted with Tet alone or combined with HGF treatment (Figure 1A, lanes 5 and 6; Additional file 1, lanes 8-9). Concerning T24-Met3 cells, expression of c-Met was suppressed 24 h after treatment with Tet (Figure 1B, lane 2). Together, auto-phosphorylation occurred when c-Met was over-expressed, and HGF treatment further enhanced the phosphorylation of c-Met. The results demonstrate a successful in vitro model in modulating the expression of c-Met using Tet-off system. Page 4 of 12 Expression and functional association of c-Met with Axl and PDGFR-a in vitro To identify the novel interaction partners of c-Met, NIH-Met5 cells were first treated with Tet for 24 h, and then cultured in the absence of Tet for an addi- tional 4 and 7 days (Figure 2A), respectively. Total RNA was extracted and subjected to screening using a cDNA microarray as previously described [35]. Among 192 RTKs, a total of 8 genes were positively correlated with c-Met over-expression, including Axl, PDGFR-a, PDGFR-b, ERBB2, ERBB3, MST1R, TIE1 and TIE2 (table 1). One of these candidate genes-MST1R was recently reported in our laboratory [6]. In addition, co- expression of c-Met with Axl and/or PDGFR-a was also detected in our pilot molecular profiling of RTKs in human bladder cancer cells in vitro [16]. As a result, both Axl and PDGFR-a were chosen for subse- quent analysis. The comparable expression patterns of c-Met, Axl and PDGFR-a at RNA level were shown in Figure 2A. Figure 1 c-Met expression in two stable cell lines in the presence or absence of Tet and/or HGF. The total cell lysate (100 μg) from the treated cell lines was initially precipitated by the phosphorylated tyrosine antibody (p-Tyr), and then blotted with c-Met antibody to evaluate the phosphorylation of c-Met. The expression level of c-Met protein was measured by Western blotting. b-actin was used as an internal control. The intensity of each band in the gel was quantified by densitometry analysis using VisionWorks™ LS image acquisition and analysis software (UVP, USA) and labeled under each band. A: Both NIH/3T3 and NIH-Met5 cells were treated with or without Tet for 24 h, and then replaced with HGF (30 ng/ml) for 10 min. B: The stable human bladder cancer cell line T24-Met3 was maintained in the medium in the presence or absence of Tet for 24 h.