Modulating Antiviral Responses via RIP Knockdown

For poly (I:C) transfection, cells were transfected at ~70% confluence with the final concentration of 50 μg/ml poly (I:C) using Lipfectamine 2000 (Invitrogen) following the manufacturer’s protocol. For gene knockdown, 150 nmol/L of double-stranded RIP1 siRNA oligonucleotides 5′-UGCUCUUCAUUAUUCAGUUUGCUCCAC-3′, RIP3 siRNA oligonucleotides 5′-UAACUUGACGCACGACAUCAGGCUG-3′, or scrambled siRNA oligonucleotides 5′-UUCUCCGAACGUGUCACGU-3′26 (link) were chemically synthesized (Beijing AuGCT DNA-SYN Biotechnology, China), and cathepsin D siRNA was purchased from Santa Cruz Biotechnology. The siRNA was transfected using Lipfectamine 2000. Quantitative PCR was performed to identify the knockdown efficacy using the following primers: RIP1, 5′-TGGGAAAGCACTGGAAAAAC-3′ and 5′-GTCGATCCTGGAACACTGGT-3′; RIP3, 5′-TTTGGCCTGTCCACATTTCAG-3′ and 5′-GGTTGGCAACTCAACTTCTCTT-3′; Cathepsin D, 5′-TGCTCAAGAACTACATGGACGC-3′ and 5′-CGAAGACGACTGTGAAGCACT-3′. At 36 hours post-transfection, cells were infected with ADV (MOI = 0.1) in the presence or absence of dsCARE. The antiviral efficacy of dsCARE was examined by fluorescent microscopy.