Pstat3 luc

To measure AP-1, NF-κB, NRF2, p53 and STAT3 transcriptional activity, Caco-2 or HCT-15 colon cancer cells were seeded in 12-well plates (4.0 × 10⁵ cells/well). After a 24-h incubation, the cells were transiently transfected with 1 μg/well of the pAP1-Luc, pNF-κB-Luc, pNRF2/ARE-Luc, pP53-Luc, pSTAT3-Luc or pTA-Luc (Signosis Inc., Santa Clara, CA, USA) reporter plasmid and 100 ng/well pGL4.73 [hRluc/SV40] control plasmid (Promega, Madison, WI, USA) using Polyethylenimine Max MW 40,000 (PolyScience, Warrington, PA, USA); transfected cells were cultured for an additional 24 h, treated with the test agents (Figure 1) for 24 h and firefly and Renilla luciferase activities were determined using the Bright GLO and Renilla GLO Systems (Promega), respectively. In the case of the cytokine mixture experiment, transfected cells were cultured in the presence of 50 ng/mL TNFα (Perotec, NJ, USA), 5 ng/mL IL-1β and 50 ng/mL EGF (Miltenyi Biotec Inc., CA, USA) for 24 h and 48 h after 30 min incubation with irsogladine maleate. The basal luciferase activity of NF-κB in the control was set as 1.0. The percentage luciferase activity with each treatment was calculated from the data of triplicate wells, with values normalized by those of the Renilla luciferase activity. The data are expressed as the means ± SD (n = 4).

To measure the AP-1, HIF, HSF, NF-κB, NRF2, p53, and STAT3 transcriptional activity, Caco-2 colon cancer cells were seeded in 96-well plates (1.0 × 10⁵ cells/well). After a 24 h incubation period, the cells were transiently transfected with 100 ng/well of pAP1-Luc, pNF-κB-Luc, pNRF2/ARE-Luc, pp53-Luc, pSTAT3-Luc, or pTA-Luc (Signosis Inc., Santa Clara, CA, USA) reporter plasmid and 10 ng/well pGL4.73 [hRluc/SV40] control plasmid (Promega, Madison, WI, USA) using Lipofectamine 2000 Transfection Reagent (Life Technologies, Inc., Gaithersburg, MD, USA). Transfected cells were cultured for an additional 8 h and treated with 500 µM acetazolamide for 24 h. Then, the firefly and Renilla luciferase activities were determined using the Bright GLO and Renilla GLO Systems (Promega), respectively. The ratio of luciferase activity with each treatment was calculated from the data of triplicate wells with values normalized by the Renilla luciferase activity. In HCT-15 or SW48 cells, the NRF2 activity was measured using the same procedure. The data are expressed as the means ± SD.

Chinese hamster ovary (CHO) cells were suspended in Ham’s F12 Nutrient Mixture (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (Biowest, Nuayer, France), 1% of the HT supplement (Thermo Fisher Scientific), 100 U/mL penicillin, and 100 μg/mL streptomycin mixture (Nacalai Tesque, Kyoto, Japan) and cultured in a humidified incubator at 37 °C and 5% CO2. The lepr open reading frame sequence of the chub mackerel (GenBank: KP635451) was digested with the restriction enzymes NheI (NIPPON Genetics, Tokyo, Japan) and BamHI (NIPPON Genetics) and then subcloned into a pcDNA3.1 (+) expression vector (Thermo Fisher Scientific). Before reaching confluence, the cells were seeded into a 6-well plate at 6 × 10⁵ cells/well. After 24 h, the expression vector (1 μg/well), pSTAT3-Luc (1 μg/well; Signosis, Santa Clara, CA, USA), and pRL-TK (8 ng/well; Promega, Madison, WI, USA) were transfected using the X-treme GENE HP DNA Transfection Reagent (Roche Diagnostics, Basel, Switzerland). The cells were harvested at 24 h after transfection and plated in 96-well plates at 3 × 10⁴ cells/well. After 24 h, the culture medium was changed to a serum-free medium containing silkworm pupae or E. coli-produced leptin (10⁻¹² to 10⁻⁷ M) and then incubated for 3 h. The luciferase activity in the cell lysate was measured using the Dual-Luciferase^® Reporter Assay System (Promega).