shRNA constructions for lentivirus package.

The target sequences of shRNAs for human TLR3 and IRF1 genes and the sequence of the negative-control shRNA were as follows: shTLR3-1#, 5′-GCTTGGATGTAGGATTTAACA-3′; shTLR3-2#, 5′-GGTAACGATTCCTTTGCTTGG-3′; shTLR3-3#, 5′-GGAGCACCTTAACATGGAAGA-3′; shIRF1-1#, 5′-GCCAGATATCGAGGAGGTGAA-3′; shIRF1-2#, 5′-GGGATATTGGGCTGAGTCTAC-3′; shIRF1-3#, 5′-GCGTGTCTTCACAGATCTGAA-3′; shNC, 5′-CAACAAGATGAAGAGCACCAA-3′.

HEK293T cells were transfected with the pLKO.1 vector encoding the negative-control or gene-targeting shRNAs, psPAX2, and pMD2.G at the ratio of 4:3:1 using Lipofectamine 2000. Forty-eight hours later, the lentivirus from supernatants was collected to infect FHC or HT29 cells. After an additional 48 h of culture, cells were selected by 1.5 μg/ml puromycin (Sigma-Aldrich, St. Louis, MO, USA). The efficiency of shRNAs to knock down the target genes was evaluated by immunoblot analysis.