Plvx puro

Short hairpin RNA (shRNA) oligos targeting TRIM46 (shTRIM46-1, 5′-GGTGAGGATATGCAGACCT-3′; shTRIM46-2, 5′-GATATGCAGACCTTCACTT-3′) were ligated into pLKO.1 (Addgene, Watertown, MA). Wild type human TRIM46 (TRIM46 WT), RING mutant human TRIM46 (TRIM46 WT) [20 (link)], and PHLPP2 was synthesized by Genwiz (Suzhou, China) and inserted into pLVX-puro (Addgene, Watertown, MA). Virus of shTRIM46, control shRNA (shNC), pLVX-TRIM46 (oeTRIM46), pLVX-PHLPP2 (oePHLPP2), or pLVX-puro (Vector) was produced in 293T cells.

For the construction of the expression plasmid for GGCT/MRPL9, the GGCT/MRPL9DE CDS sequence was amplified from human cDNA and ligated into the pLVX-puro (Addgene, Watertown, MA, USA) vector. The procedure followed the instructions for the cloning kit and EcoRI restriction endonuclease sites were used (10911ES20, Yeasen, Shanghai, China). The plasmid of shGGCT/shMRPL9 was synthesized in Tsingke Biotechnology Co., Ltd. (Tsingke Biotechnology, Beijing, China). The PCR amplification primers and sh-RNA sequences mentioned above are provided in Supplementary Table S1. For lentivirus packaging, PEI (40820ES04, Yeasen, Shanghai, China). was used to transfect the target plasmid, pCMV-VSV-G (Beyotime, Shanghai, China) and pCAG-dR8.9 (Beyotime, Shanghai, China) in 293T cells (according to the ratio of target plasmid: pCMV-VSV-G: pCAG-dR8.9 = 4:3:1). The medium was replaced with fresh medium 6 h after transfection, and the virus stock solution was collected 48 h after transfection. For stably transfected cell lines, PTC cells were infected with LV-GGCT, LV-MRPL9, LV-sh-GGCT, LV-sh-MRPL9, LV-pLVX, LV-pLKO.1, then cells were treated with 1 ug/mL puromycin (Meilunbio, Dalian, China).

FLAG-HA-pcDNA3.1 (#52535) was purchased from Addgene and renamed pcDNA here. Based on this, we constructed plasmids expressing MOV10 and IKKε with Flag-HA tags. The ICP0-WT plasmid (pRS-1) and ICP0-M138 plasmids were previously described [44 (link),65 (link)]. To construct ICP0no3’UTR plasmid, the sequence between MluI and stop codon of pRS-1 was amplified and inserted between MluI and HindIII sites of pRS-1. As for ICP0nointron plasmid, ICP0 CDS fragment was amplified and then inserted between NcoI and MluI sites of pRS-1. To construct plasmids expressing truncated MOV10 mutants, we amplified the truncated Mov10 gene sequences by PCR and cloned them into the FLAG-HA-pcDNA3.1 vector using the Site-Directed Mutagenesis kit (#E0552S) purchased from New England Biolabs. Human MOV10 expressing sequences were amplified and inserted between the BamHI and XbaI restriction sites of pLVX-puro (Addgene) plasmid to construct pLVX-puroMOV10 plasmid for lentivirus production. Plasmids expressing MOV10 N- terminus (amino acids 1–495) and MOV10 C-terminus (amino acids 496–1003) were generously provided by Professor Zhiwei Wu (Nanjing University) [49 (link)]. Renilla luciferase plasmid and IFN-β promoter reporter plasmid were kindly provided by Chunfu Zheng at Fujian Medical University. The sequences of all primers used are listed in S2 Table.

The lentivirus pLVX-Puro (Addgene) was obtained from DesignGene Biotechnology (Shanghai, China) and used to clone shRNA sequences. The vectors were designated Lv-eIF4A1 (eIF4A1-1, 5′-CACACTGGACTAGTGGATCCCGCCACCATGTCTGCGAGCCAGGATTCCC-3′; eIF4A1-2, 5′-AGTCACTTAAGCTTGGTACGATGAGGTCAGCAACATTGAGG-3′), Lv-sh-c-MYC (c-MYC-1, 5′-GCTTCACCAACAGGAACTATG-3′; c-MYC-2, 5′-GCTTGTACCTGCAGGATCTGA-3′; c-MYC-3, 5’-GGAAACGACGAGAACAGTTGA-3′) and Lv-sh-control (empty vector). The lentivirus plasmid and packaging plasmids were transfected into pancreatic cancer cells with transfection reagent (Lipofectamine®3000, Thermo Fisher Scientific) in OPTI-MEM media (Invitrogen, MA, USA). Lentiviral infection of the target cells was performed in cell culture medium containing 5 μg/ml polybrene (Sigma H9268), and infected cells were selected with 2.5 μg/ml puromycin for follow-up experiments.