Induction analysis of TNF-α by HCV at early time points.

<p>(A) The time-course analysis of TNF-α induction by HCV. Huh7 cells were infected with HCV (MOI = 2) for 0, 1, 2, 4, 8, and 24 hours and then lysed for the isolation of total RNA, which was analyzed for TNF-α and actin RNAs by semi-quantitative RT-PCR. (B) The induction of TNF-α by HCV using the MOI of 0.25 and 1 was analyzed by qRT-PCR. (C) Huh7 cells were infected with HCV (MOI = 1) that had been pre-incubated with the isotype control antibody or the anti-E2 antibody at 4°C for 1 hour [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004937#ppat.1004937.ref013" target="_blank">13</a>]. The total cellular RNA was isolated at 2 hours post-infection for qRT-PCR analysis of TNF-α RNA. TNF-α RNA induced by HCV that was treated with the isotype antibody control was arbitrarily defined as 100%. (D) Effect of actinomycin D on TNF-α induction by HCV. Huh7 cells were pretreated with DMSO or actinomycin D (5 μg/ ml) for 1 hour. Cells were then infected with HCV (MOI = 1) for 2 hours. Total cellular RNA was isolated for qRT-PCR analysis to assess TNF-α induction. TNF-α RNA induced by HCV in cells treated with DMSO was arbitrarily defined as 100%. (E) UV-inactivation of HCV. HCV (MOI = 1), with or without UV-irradiation for 5 minutes, was used to infect Huh7 cells. Cells were lysed at 2 hours and 24 hours post-infection for qRT-PCR analysis of TNF-α RNA. TNF-α RNA induced by HCV at 2 hours post-infection without UV treatment was arbitrarily defined as 100%. *, <i>p</i><0.05.