Bright glo reagent

The CDC37 promoter-luciferase reporter constructs (500+UTR and 200+UTR) [60 (link)], the overexpression constructs of heat shock factor 1 (HSF1) and dominant-negative (DN)-HSF1 [74 (link)], plasmid DNA co-transfection, and luciferase assay were described previously [60 (link),74 (link),78 (link)]. Briefly, cells were cultured in 96-well plates and a plasmid (25 ng reporter, 100 ng effector) was transfected with 0.4 µL FuGENE HD (Roche, Basel, Switzerland) per well at a cell confluence level of 50%–70%. The medium was changed at 16–20 h after transfection. At 40–48 h after transfection, 70 µL of the medium was aspirated, then 30 µL of Bright-Glo reagent (Promega, Madison, WI, USA) was added and mixed by pipetting. Cells were incubated for 5 min at 37 °C. The lysate (40 µL) was transferred to a 96-well white plate for measurement of luminescence.

To determine the effect of the cocoa extract on F. nucleatum-induced NF-κB activation, the U937 3xκB-LUC monocytes were seeded (10⁵ cells/well) in the wells of a black wall, black bottom, 96-well microplate (Greiner Bio-One North America) and were pre-incubated with non-cytotoxic concentrations of cocoa extract for 30 min. Thereafter, the monocytes were stimulated with F. nucleatum at an MOI of 100 for 6 h. Wells containing monocytes but no F. nucleatum or no cocoa extract were used as controls. A commercial inhibitor (BAY-11-7082 [25 μM], EMD Millipore Canada, Mississauga, ON, Canada) of the NF-κB signaling pathway was used as a positive control. Bright-Glo reagent (Promega Corporation, Durham, NC, USA) was used according to the manufacturer’s protocol to measure luciferase activity and determine NF-κB activation. Luminescence was monitored using a Synergy 2 microplate reader. Assays were performed in triplicate in two independent experiments.

The human monoblastic leukemia cell line U937 3xκ B-LUC, a subclone of the U937 cell line stably transfected with a luciferase gene linked to a promoter of three NF-κB-binding sites [28 (link)], was used to investigate the effect of eriodictyol on P. gingivalis-induced activation of the NF-κB signaling pathway. These cells were cultivated in RPMI-10% FBS, 100 μg/mL of penicillin G/streptomycin, and 75 μg/mL of hygromycin B at 37°C in a humidified incubator with a 5% CO₂ atmosphere. The U937 3xκB-LUC monocytes were seeded (10⁵ cells/well) in the wells of black wall, black bottom, 96-well microplates (Greiner Bio-One North America) and were pre-incubated with eriodictyol (80, 160, or 320 μM) for 30 min. The monocytes were then stimulated with P. gingivalis at an MOI of 100 for 6 h. Wells containing monocytes without bacteria or eriodictyol were used as controls. Bright-Glo reagent (Promega Corporation, Durham, NC, USA) was used according to the manufacturer's protocol to measure luciferase activity and determine NF-κB activation. Luminescence was monitored using a Synergy 2 microplate reader. Assays were carried out in triplicate in three independent experiments, and the means ± standard deviations were calculated.