NF-κB Transcriptional Activity Measurement

Luciferase assay was performed to measure the transcriptional activity of the NF-κB in HCs under pepsin exposure at each pH point compared to control. A NF-κB reporter Vector [pGL4.32(luc2P/NF-κB-RE/Hygro)], a control vector [pGL4.27(luc2P/minP/Hygro)], a Lipofectamine^® 2000 (Invitrogen™, Thermo Fisher Scientific, Waltham, MA, USA) and a firefly Luciferase Assay system (Promega Corporation, Madison, WI, USA) were used, according to manufacturer’s procedure, as previously described [56 (link),57 (link),58 (link)]. The treatment was performed 24 h after transfection. The cells were treated with pepsin (1 mg/mL) at pH 5.0, 6.0 and 7.0 and controls, for 15 min and then media were replaced with serum-free medium. After 4–6 h incubation we measured luminescence using a luminometer and Gen5 software (Synergy1, BIOTEK; Gen5^TM software, BioTek Instruments Inc., Winooski, VT, USA). NF-κB activity was expressed as ratios of mean values of NF-κB reporter (NF-κBLuc2P) against mean values of control (Luc2P) for each condition (pepsin at pH 5.0, 6.0, 7.0 and control). Triplicate assays were performed for each treatment condition.