Pgl4.75 hrluc cmv plasmid

Luciferase assays were performed to determine the NF-κB activity in response to poly(dA:dT) treatment. The HPV-KER cell line was transfected with the NF-κB reporter construct vector pNF-κB-luc Cis-Reporter Plasmid (Stratagene, La Jolla, CA, USA) and the pGL4.75 [hRluc/CMV] plasmid (Promega, Madison, WI, USA) with the use of the X-tremeGene9 transfection reagent. The treated cells were washed twice with PBS, lysed with passive lysis buffer (Biotium, Hayward, CA, USA) and the luciferase activities in the lysates were measured using the Firefly & Renilla Dual Luciferase Assay Kit (Biotium) and Thermo Luminoskan Ascent (Thermo Scientific, Rockford, IL, USA), according to the manufacturer’s instructions. All samples were measured three times and the luciferase activity derived from the NF-κB-luc plasmid was normalized to the activity of the Renilla luciferase activity from pGL4.75 [hRluc/CMV] plasmid.

The HEK293 cell line (Merck, Darmstadt, Germany) was maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Lonza, Basel, Switzerland), 1% L-glutamine (Lonza) and 1% antimycotic–antibiotic solution (Lonza) at 37 °C in a humidified atmosphere with 5% CO₂.
For transfection, cells were seeded into 12-well plates at a density of 300,000 cells/mL in full medium. After 48 h, medium was changed to FBS-free medium, and cells were co-transfected with the AP1–LUC cis-reporter plasmid, the pGL4.75 (hRluc/CMV) plasmid (Promega), which was used as internal control, and 1µg pcDNA3.1+/C-(K)DYK vector (GenScript) carrying the WT or mutant CCDC88C cDNA sequences. Transfection was carried out with the Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. Mock-transfection by transfection reagent and cotransfection of luciferase plasmids with the empty pcDNA3.1(+) plasmid served as control. Twenty-four hours after transfection, cell samples were collected for extraction of proteins, which were subjected to luciferase activity measurement or to TUNEL assay.