Geneporter 3000

The Adamts-1 promoter region, ranging from − 1705 to + 90, was amplified from mouse genomic DNA by PCR with Forward (5′-ACT GTG GAT GTC AGT GAG AGC-3′) and Reverse (5′-GCT GCT TTC TAG CGA GTG CAA-3′) primers. The amplified PCR product was cloned into the pGL4.10-basic reporter, and the resulting plasmid was designated as pGL4. 10/Adamts-1 (− 1705/+ 90). Reporter vectors containing the Adamts-1 promoter region from − 1705/− 978 or − 977/+ 90 were generated using pGL4.10/Adamts-1. Mutant promoter constructs (− 1151/− 1134 mut) were produced using the EZ change™ site-directed mutagenesis kit (Enzynomics, Seoul, Korea). In 12-well plates, 293 T cells were seeded and transfected with 2 μg of the Adamts-1 promoter-reporter construct and EGR1 expression vector (pIRES dsRED2/EGR1) using Gene Porter 3000 (Genlantis, San Diego, CA, USA) transfection reagents, following the manufacturer’s instructions. A pRL-null plasmid (50 ng) encoding Renilla luciferase was incorporated in all samples to monitor the transfection efficiency. After 48 h, the firefly and Renilla luciferase activities were measured sequentially from a single sample using the Dual-Glo™ Luciferase Assay System (Promega, Madison, WI, USA) using an illuminometer.

HCEn cells were transfected with an IRF7-expressing plasmids using a CMV promoter (pCMV6 backbone, OriGene, Rockville, MD) with gene porter 3000 (Genlantis, San Diego, CA). The extracted proteins were confirmed by western blot analyses using an anti-IRF7 antibody (Santa Cruz Biotechnology, Dallas, TX) and HRP-conjugated secondary antibody (Cell Signaling, Danvers, MA).
For siRNA transfection, HCEn cells were transfected with IRF7 siRNA (Santa Cruz Biotechnology, Dallas, TX) using RNAifect (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.

The coding sequence of the monkey HO-1 gene (XM028827760) was synthesized and ligated into a 3.1 (+) vector (Invitrogen, Carlsbad, CA, USA). pcDNA3.1 (+) (pcDNA3.1 (+)/mkHO-1) inserted with the monkey HO-1 gene or the empty vector control pcDNA3.1 (+) was subsequently transfected into FRhK-4 cells using GenePORTER 3000 (Genlantis, San Diego, CA, USA). We incubated 70% confluent FRhK-4 cells in a 6-well plate with a mixture of 4 μg DNA vector and GenePORTER 3000 reagent in serum-free medium for 4 h. Following incubation, FBS was added to the medium at 10%.