PD98059, Wortmannin, Lysis buffer was purchased from Beyotime, China. Primary antibodies have been obtained from Santa Cruz Biotechnology, CA, USA. Rabbit anti Nrf2 was bought from Abcam, USA. Major antibodies had been bought from Cell Signaling Technology, MA, USA. Goat anti rabbit IgG and goat anti mouse IgG antibodies had been bought from LI COR, Lincoln, NE, USA. Rabbit anti Goat IgG was obtained from KPL, Gaithersbhrg, MD, USA. Monoclonal mouse anti glyceraldehyde 3 phosphate dehydrogease was obtained from KangChen, China. Cell lines and cell culture Human epithelial colorectal adenocarcinoma cell line Caco two, Human colon adenocarcinoma grade II cell line HT 29, human liver carcinoma cell line HepG2,Human Embryonic Kidney 293 cell HEK 293 had been bought from Cell Bank of Shanghai Institute of Biochemistry and Cell Biology.
Cells were cultured in DMEM medium, MEM medium, McCOYs 5A supplemented with 10% inhibitor MP-470 fetal bo vine serum, one hundred U ml penicillin and one hundred ug ml streptomycin. All cultures have been maintained in a humidified atmosphere with 5% CO2 at 37 C. Transient transfection and analysis of luciferase reporter gene activity We made use of the luciferase reporter assay to investigate the Nrf2 mediated transcriptional activity of Nrf2. Firstly, 8 copies of antioxidant responsive element luciferase reporter plasmids were generated utilizing the pGL3 pro moter vector. Right after the plasmids have been gen erated, the DNA sequence of the inserts was verified. The Dual Luciferase Reporter Assay System was utilized to decide reporter gene activity in transiently transfected cells.
Transient transfection was performed i thought about this in 96 properly plates at a cell density of 50% 70% confluence per effectively. Then the 8 ? ARE pGL3 plasmid had been co transfected together with the pRL TK plasmid, encoding Renilla lucifer ase as an internal control for transfection efficiency for 24 h making use of Lipofectamine 2000 in line with the companies instructions. Just after transfection, cells have been treated with test samples for indi cated time, then cell lysates have been ready for assess ment of luciferase activity. Fire fly and Renilla luciferase activities have been measured applying a luminometer based on the manu facturers guidelines. Relative fire fly luciferase activity was normalized to Renilla luciferase activity and activity was expressed as fold induction immediately after treatment with com pounds compared with automobile handle. Cell viability assay Cell viability was determined working with the MTT assay. Briefly, cells in logarithmic phase were seeded in the density of 70 80% confluence per nicely in 96 nicely plates at 37 C with 5% CO2 for overnight incubation and treated with appropriate concentrations of test samples for the indicated instances. Following treatment, ten ul of 5 mg ml MTT was added and also the cells have been incubated for four h at 37 C.