Firefly luciferase reagent

Dual luciferase assays were performed with the dual luciferase reporter assay system using the firefly luciferase reagent (LARII) and the Renilla luciferase reagent with firefly quenching (Stop & Glo) (Promega). All reagents were prepared as described by the manufacturer. Protoplasts were re-suspended in 50 μl 1X passive lysis buffer and incubated on ice for 15 min. The lysates were then centrifuged for 15 min at maximum speed at 4°C. Ten μl of undiluted supernatants were used to monitor the bioluminescence using a Centro SX3 luminometer (Berthold Technologies). Statistical significance was examined by One-Way analysis of the variance (ANOVA) and the Tukey's HSD post-hoc test with the level of significance set at 5%.

500 ng Top-Flash plasmid mixed with 200 ng CAG-CTNNB1-IRES-Neo plasmid and 500 ng pCMV6-GATA4 plasmid or 500 ng Empty Vector was transfected to A549 cell seeded in 12-well plate via VigoFect reagent. Both the treatment had three replicates. After 48 h culture, A549 cells were trypsinized and counted. 100 μL culture medium within 20,000 A549 cells was mixed with 100 μL Firefly Luciferase reagent (E1910, Promega, Madison, WI, USA) to measure the value of luminescence. For evaluation the effect of Wnt pathway inhibitors in Top-Flash assay, three inhibitors were added to the A549 cells post 24 h transfection for another 48 h incubation until the day of measurement.

Keratinocyte activation by the Keap1/Nrf2-ARE (antioxidant response element) pathway was measured by the KeratinoSens^TM assay [48 ]. The assay uses an antioxidant response element (ARE)-coupled luciferase to sense Nrf2 activation. The KeratinoSens™ cell line was obtained from Givaudan (Vernier, Switzerland). The cells were cultured in DMEM supplemented with Glutamax^TM, Fetal Calf Serum (FCS) 9.1%, and Geneticin^TM (500 μg/mL) at 37 °C in an atmosphere of 5% CO₂ and 95% humidity. Cells were seeded on 96-well plates until reaching 80% confluency. Cells were then incubated with obacunone for 48 h in antibiotic-free DMEM. At the end of the incubation period, cells were washed and lysed for 20 min. Then, Promega firefly luciferase reagent was added, and the luminescence was immediately measured on a Lumistar plate reader (Lumistar Omega, BMG Labtech). An increase in luciferase activity in sample-treated cells was calculated in comparison to DMSO-treated cells (negative control) and expressed as fold luciferase activity induction (Imax). Following the manufacturer’s instructions, activation of the Nrf2 pathway was considered positive when the Imax was equal to or higher than 1.5 and statistically significant compared to the negative control.