Easy mutagenesis system

Expression vectors for GSNOR, TBK1, and IRF3 truncate (IRF3-5D [27 (link),28 (link)]) were purchased from the Miaoling Plasmid Sharing Platform (MLPSP). Site-directed mutants of GSNOR (p.R115D) and TBK1 (p.C423S, p.C426S, and p.C471S) were constructed using the Easy Mutagenesis System (Beijing TransGen Biotech) according to the manufacturer's protocols. All constructs were confirmed by DNA sequencing. The transient transfection of vectors was performed using Lipofectamine 3000 (Invitrogen, L3000015).

Besides the initial residue Met¹, the N-terminal sensor region of RpfC contains 21 residues, with two of them being Ala. To conduct alanine-scanning mutagenesis, full-length rpfC coding sequence was amplified by PCR and inserted into a pGEM T-easy vector (Promega, USA), and Easy Mutagenesis System (TransGen Biotech, China) was used to construct point mutations according to the manufactory’s manual. Coding sequences of 19 non-Ala residues were mutated into Ala, respectively, and Ala¹⁶ and Ala²¹ were mutated into Val, respectively. The point mutation was confirmed by sequencing. These inserts with corresponding point mutations were cut by restriction enzymes, purified, and ligated into broad host vector pHM1 [48 (link)]. Their expressions were under the control of a PlacZ promoter. Recombinant vectors were then electroporated into ΔrpfC or ΔrpfCΔrpfF mutants as needed. Primers used to create these mutants are listed in S2 Table.

The 3′ untranslated regions (UTRs) of the human CHN1 gene (NM_001025201.2) were amplified by polymerase chain reaction (PCR) from human genomic DNA, cloned into the SbfI and NheI site of the pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega), checked for orientation, and sequenced; the resulting plasmid was named pmirGLO-CHN1-wt. PCR primers used to amplify the CHN1 3’UTR are shown in Table 2. Site-directed mutagenesis of the miR-205 target site in the CHN1 3’UTR was carried out using an Easy Mutagenesis System (Transgen, China), with pmirGLO-CHN1-wt as a template; the resulting plasmid was named pmirGLO-CHN1-mut.
Next, 5 × 10⁴ cells were seeded in each well of a 48-well plate at 24 h before transfection. For reporter assays, the cells were transiently cotransfected with 0.25 μg wild-type (WT) or mutant reporter plasmid and 7.5 pmol NC or miR-205 mimic using Lipofectamine 2000. At 48 h after cotransfection, Firefly and Renilla luciferase activities were measured consecutively using Dual Luciferase Assays (Promega) according to the manufacturer’s instructions. Three independent experiments were performed.

Human epithelial HeLa, HEK293T and MDA-MB-231 cell lines were purchased from the American Type Culture Collection (ATCC) and were cultured in DMEM (GIBCO) supplemented with 10% foetal bovine serum (FBS, Hyclone) and 1% penicillin/streptomycin at 37 °C under 5% CO₂. The cell lines have been tested free for Mycoplasma contamination. Hypoxic conditions were created by culturing cells in the hypoxia chamber (Billups-Rothenberg) flushed with 1% O₂, 5% CO₂ and 94% N₂ mixture gas. Human recombinant TGF-β1 was purchased from Peprotech. Plasmids were transfected with Lipo2000 according to the manufacturer's instruction. SB431542 (S1067) was purchased from Selleck Chemicals. Cytochalasin D was purchased from Sigma. Expression plasmids of Siah2 and Zyxin were generated by PCR and cloned in pFlag-CMV-4, pGEX-4T-1 or pEF1/V5-His expression vectors. Myc-Lats2 plasmid was constructed by insertion of Lats2 cDNA in frame into the pcDNA3.0 vector. All Siah2 and Zyxin mutant constructs were created using the Easy Mutagenesis System (TransGen Biotech). All the plasmids were confirmed by DNA sequencing.

The cDNA encoding human DDX3 was cloned into pLVX-AcGFP1-N1 and pET28a for cell transfection and expression, respectively. The different truncated DDX3 mutants were amplified from the cDNA of human DLD1 cells and then cloned into pLVX-AcGFP1-N1, named pLVX-AcGFP1-K255A, -R287A, -S290A, and R294A were generated by the Easy Mutagenesis System of Transgen Biotech (Beijing, China) and verified by DNA sequence analysis.

cDNA was synthesized with reverse transcriptase M-MLV (Takara) by oligo(dT) priming of total RNA from a normal human thyroid gland. The DUOX2 and DUOXA2 open reading frames were amplified using native Pfu polymerase and cloned into pcDNA3.1 (Invitrogen). The primers for DUOX2 were 5′-GGGGTACCCCATGCTCCGTGCAAGACCAGAG-3′ and 5′-GCTCTAGAGCTCAGAAGTTCTCATAGTGGTGC-3′ and the primers for DUOXA2 were 5′-GGGGTACCCCATGACCCTGTGGAACGGCGTA-3′ and 5′-GCTCTAGAGCTCACAGGTTAGTGGTGATACA-3′. The two novel DUOX2 mutations p.R354W and p.A1206T were introduced into the DUOX2 expression vector by site-directed mutagenesis (Easy mutagenesis system, TransGen) using the following primers: p.R354W: F 5′-TGCCAGCTGTCATTTCTGGAAGGTCCT-3′ p.R354W:R 5′-AGAAATGACAGCTGGCATTTCTCATGTAG-3′ and p.A1206T: F5′-ATCATGTATGTCTTCACCTCCCACCA-3′ p.A120 6T:R5′-TGAAGACATACATGATGGCCAGGACC-3′. All constructs were confirmed by bidirectional DNA sequencing.