Pegfp c1

Expression vectors encoding FLAG-tagged KAT8 were constructed by PCR cloning into pCMV-FLAG (Invitrogen, USA) eukaryotic expression vector. The ORF frame of CiKAT8, CiIRF3 and CiIRF7 were separately inserted into pEGFP-C1 (Promega, USA) vector for co-IP, subcellular localization and DNA pull-down assays. The amino acid truncations KAT8-(△1-151), KAT8-(△151-264) and KAT8-(△264-487) were generated by PCR-based amplification and constructed into the pCMV-FLAG, pEGFP-C1 vector respectively. The ORF of CiKAT8, CiIRF3 and CiIRF7 were separately inserted into pcDNA3.1 (+) vector (Invitrogen, USA) for over-expression experiments. IFN 1 and ISG15 promoter luciferase reporter plasmids were prepared in our lab (37 (link)); pISRE-TA-luc (beyotime, China) was used in luciferase assays. Each construction was confirmed by DNA sequencing. The primers for plasmid construction were given in Table 1.
For transient transfection of poly(I:C), B-DNA or Z-DNA into CIK cells, Lipofectamine 2000 (Invitrogen, USA) was used according to the manufacturer’s instructions. Empty plasmid or various expression plasmids were transfected in CIK cells and CO cells using Lipo8000^™ transfection reagent (beyotime, China) according to the manufacturer’s protocol.

The 3′UTR of Atg12 (NM_026217) including the predicted sites (α:1407 site, β:1895 site, γ:1407 site-1895 site) for miR-30a-3p seizing was obtained by PCR. Then, the Atg12 3′UTR was cloned into the pMIR-REPORT vector (Thermo Fisher Scientific, Waltham, MA, USA). The mutational site was obtained by site-directed mutagenesis. miR-30a-3p or control together with these luciferase vectors were co-transfected into 293T cells by a Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) protocol in triplicate. Cells were lysed 48 h after transfection; then, the Dual-Light Luciferase and β-Galactosidase Reporter Gene Assay System (Thermo Fisher Scientific, Waltham, MA, USA) was used to assay the luciferase activity according to the manufacturer’s protocols. Mmu-miR-30a-3p (ENSMUSG00000065405) was obtained by PCR. pEGFP-miR30a-3p vector was constructed with pEGFP-C1 (Promega, Madison, WI, USA) to stabilize expression of miR-30a-3p, and pEGFP-miR30a-3p was verified by restriction enzyme and sequencing.

Genomic DNA was extracted from cells by proteinase K digestion and phenol:chloroform extraction followed by ethanol precipitation and resuspension overnight in TE (pH 8.0). Genomic DNA concentration was quantified with UV spectrophotometer (NanoDrop) and diluted to 16 ng/μl for qPCR. For standard curve, a series of mixtures in which the number of pEGFP-C1 (Promega) plasmid molecules ranged from 128 to 1024 copies per HeLa genome were prepared using HeLa genomic DNA. 32 ng of genomic DNA from different cell lines or copy number standards were subjected to quantitative PCR. Quantitative PCR was performed on Roche LightCycler 2.0 using qPCR Core kit for SYBR® Green I No ROX (Eurogentec). qPCR reactions with copy number standards were performed in duplicate. qPCR reactions with cell line genomic DNAs were performed in triplicate. Melting curve analysis was carried out at the end of cycling to confirm amplification of a single PCR product. Following EGFP and human TRKB (genomic control) specific PCR primer sets were used: EGFP sense 5′- CAG AAG AAC GGC ATC AAG GTG-3′, antisense 5′- TGG GTG CTC AGG TAG TGG TTG -3′; TRKB sense 5′- CAC AGG GCT CCT TAA GGA TAA C -3′, antisense 5′- GCA CAG TGA GGT TGA CAG AAT C-3′. Copy number estimates were calculated with qBASEPlus 2.6 software (Biogazelle) using EGFP as target and TRKB as reference.

DU145 prostate cancer cells were plated in 24-well plates (1×10⁵ per well) for 24 h and co-transfected with 300 ng of reporter constructs (pGL3-B-miR-101-L, pGL3-B-miR-101-S, pGL3-B-miR-101-WBS, pGL3-B-miR-101-MBS or pGL3-Basic), 300 ng of pEGFP-C1-AR or pEGFP-C1 plasmids and 10 ng of pRLSV40 (Promega, San Luis Obispo, CA, USA) using Lipofectamine 2000. After transfection, the medium was replaced with fresh medium containing R1881 (1 nM) and harvested 24 h later. Luciferase activities were assayed using the dual-luciferase reporter assay system (Promega, San Luis Obispo, CA, USA) in a Turner TD20/20 luminometer and normalized to Renilla luciferase activity.

hFOB 1.19 cells seeded in 12-well plates at 50% confluence were transfected with 0.8 μg of RANKL promoter reporter plasmid DNA plus 0.2 μg of internal control pEGFP-C1 (Promega) plasmid DNA using the TransIT-2020 transfection reagent (Mirus). Cells were harvested 72 h after transfection and subsequently lysed with Passive Lysis Buffer (Promega). Bright-Glo Luciferase Assay reagent (Promega) was used to determine luciferase activity and GFP fluorescence values. The relative fold of reporter activity normalized by GFP intensity represented the ratios of promoter reporter luciferase values versus the pGL4 control luciferase value (Supplemental Figure 1).