Figure 2

Generation of dsRNA in tumor cells transfected with pSIN-β. (A). RT-PCR confirmed the presence of sindbis virus nsp4 gene mRNA and its anti-sense strand in tumor cells transfected with pSIN-β. TC-1 cells were transfected with pCMV-β (pCMV) or pSin-β (pSIN), or left untreated (N/A). Total RNA was reverse transcribed into DNA with oligo dT primer or primers specific to the nsp4 gene (forward p4F or reverse p4R) before PCR amplification. This experiment was repeated twice with similar results. (B). ELISA confirmed the presence of an elevated level of dsRNA in TC-1 cells transfected with pSIN-β (n = 3). Total dsRNA was isolated from TC-1 cells transfected with pCMV-β or pSIN-β and used to coat ELISA plate. The primary Ab was the J2 anti-dsRNA IgG2a. *, p = 0.004. (C). Transfection of pSIN-β into TC-1 cells inhibited cell growth. TC-1 cells (20 000 cells/well) were transfected with the same amount (0.4 μg) of pCMV-β, pSIN-β, or pSIN-β-Δnsp (n = 4). Cell numbers were quantified using MTT assay and normalized to cells treated with sterile PBS. Data shown are mean ± S.E.M. **, at 48 and 72 h, the value of the pSIN-β were different from that of the pCMV-β and the pSIN-β-Δnsp (p < 0.05).