pIRES2-AcGFP1 vector mRNA was amplified using primers 5′-TGATCTACTTCGGCTTCGTG -3′ (left) and 5′-CACTTGTACAGCTCATCCATG C -3′ (right) and Universal Probe Library #70 (Roche Diagnostics). In addition, to further confirm the result, metastasis was assessed

based on immunohistochemical staining using anti-AcGFP1 (Clontech Laboratories) and goat polyclonal anti-cytokeratin (CK)-19 antibodies (Santa Cruz Biotechnology, Inc, Santa Cruz, CA, USA). Statistics Values are expressed as means ± SD. Groups were compared using one-way ANOVA in combination with Dunnette’s methods and paired t test. selleck screening library Values of p < 0.05 were considered significant. Results After stably transfecting SCCVII cells with murine TGFβ1 cDNA, we initially confirmed the overexpression of TGF-β1 protein by the transfectants. Using RT-PCR with primers for full-length Fulvestrant TGF-β1 or AcGFP1 gene, we confirmed the presence of two empty

vector-transfected controls (M1, M2) and three TGF-β1-transfected clones (T1, T2, T3) (Figure 1A). When levels of TGF-β1 mRNA were measured using real time PCR (Figure 1B), tumors in mice inoculated with a TGF-β1 transfectant clone showed significantly higher levels of TGF-β1 mRNA than those inoculated with a mock transfectant. In addition, when levels of TGF-β1 protein were measured in cultured cells using ELISAs (Table 1), only TDLN lysates from mice bearing a TGF-β1-expressing tumor showed high levels of TGF-β1 (Figure 2A). By contrast, serum TGF-β1 levels did not differ between mice bearing tumors that expressed TGF-β1 and those did not (Figure 2B). Figure 1 Characterization of TGF-β1 transfectant clones. TGF-β1 gene transfection was confirmed by RT-PCR and real-time RT-PCR.

A, Expression of TGF-β1 and AcGFP1 mRNA was assessed by RT-PCR. Electrophoresis gels (a and b) show the expression of TGF-β1 and AcGFP1 mRNA, respectively. M1 and M2, mock; T1, T2 and T3, TGF-β1 transfectant clone; N, negative control (SCCVII cells). B, Relative levels of murine TGF-β1 mRNA were determined by semi-quantitative real-time RT-PCR. Levels of TGF-β1 mRNA were normalized to those of β-actin mRNA and were found to be significantly higher in TGF-β1 transfectants. Table 1 Level of TGF-β1 expression in SCCVII Phospholipase D1 cells measured using an ELISA Cultured cell supernatants TGF-β1 concentration (pg/mg protein) Statistics Wild 183.31 ± 16.91   Mock transfectants     1 216.39 ± 6.33   2 213.94 ± 10.04   TGF-β1 transfectants     clone 1 541.35 ± 7.67 P < 0.01 clone 2 392.06 ± 8.65 P < 0.01 clone 3 380.12 ± 20.12 P < 0.01 Figure 2 Concentrations of TGF-β1 in tumor draining lymph nodes. A, TGF-β1 levels in tumor-draining lymph nodes (TDLNs) and the contralateral nodes (non-TDNLs) in the same mice were assessed using an ELISA. Prior to inoculation, tumor cells were transfected with either TGF-β1 gene or empty vector (mock).