Cell culture
The endometrial cancer cells, Ishikawa cells, were used in this study. SPEC2 also is an endometrial cancer cell line and maintained in our lab. Both cell lines were cultured in Medium DMEM: F12 (1:1, BIBCO) with 100µg/ml streptomycin (Life Technologies, Inc., Rockville, MD), 100 U/ml penicillin G and 10% fetal bovine serum (FBS; Gibco, Gaithersburg, MD, USA).
IHC
Total 120 endometrial tissue samples, including 10 cases of normal endometrium, 20 cases of simple hyperplasia and 90 cases of endometrial cancer, were enrolled in this study. These samples were collected from the Department of Obstetrics and Gynecology of Shanghai First Maternity and Infant Hospital, Tongji University School of Medicine, China. Samples collected in compliance with institutional review board regulations from the Medical College, Tongji University, China.
IHC analysis of Nrf2 expression pattern was performed as previously described. Briefly, firstly samples were deparaffinized in xylene and rehydrated in a graded series of ethanol, subsequently, 3.0% hydrogen peroxide was used to block the endogenous peroxidase activity. Following antigen retrieval, incubation overnight with rabbit anti-human Nrf2 primary antibodies at 4°C in a humid chamber, followed by a 60-minute incubation with biotinylated secondary antibody (Dako, Carpinteria, CA, USA). Omitted primary antibodies were used as negative controls. Expression of Nrf2 was assessed as previous described.
RNA isolation and quantitative real time PCR
Total RNA was harvested with Trizol reagent. 1µg of total RNA was reverse transcribed into cDNA using the reverse transcriptase kit. The primers used to amplification of Nrf2 and GAPDH were listed table 1. The water was served as negative control.
Western blot
The changes of the proteins were detected with western blot. The cells underwent indicated treatment were lysed and loaded to SDS-polyacrylamide gel, electrophoresed and transferred to PVDF membranes, then incubated primary antibodies and second antibodies. After final wash with PBS, the bands was detected with chemiluminescence detection system (ECL detection kit; Pierce, Rockford, IL). Each experiment was performed at least three times.
Drug treatment, Cell proliferation and Clonogenic assay
Endometrial cancer cells were treated with cisplatin, stigmasterol, or combine to both drugs with indicated doses. Cell proliferation pattern was monitored with CCK8 assay. A clonogenic assay was carried out referring previously study[26].
Plasmid transfection
Tet1 plasmid was transfected into Ishikawa cells using Lipofectamine 3000 TM (Invitrogen) according to the manufacturer’s protocol. The transfection efficiency was confirmed by western blot.
Establishment of stable cell lines
To identify the role of Nrf2 in chemotherapy resistance, the stable cell lines with high levels of Nrf2 or Keap1 were established. Ishikawa- and Spece2 derived stable cell lines, with incorporation of Nrf2 or Keap1, were established using lentivirus system as described previously. Stable Ishikawa and spece2 cells were continuously cultured in medium containing 1.5μg/ml puromycin (sigma). The efficiency of transfection was determined with western blot.
Dot blot for detection of 5hmC
Dot blot analysis was carried out as previously described. Briefly, the total DNA was extracted and loaded on nitrocellulose membranes, following blocking with 10% skimmed milk for 1 hour after bake at 80°C for 10 min, then incubation with 5hmC primary antibody (1:500 dilutions, Active Motif) overnight at 4°C. After washing the HRP-conjugated secondary antibody, the membrane was treated with ECL and scanned by a scanner. Methylene blue (MB) staining served as a loading control.
ARE luciferase activity assay
The MDA-MB-231 cell line constructed with NQO1-ARE luciferase reporter gene maintain in our lab. In order to investigate if the stigmasterol plays a role in regulating Nrf2-ARE signal pathway, the different dose of stigmasterol was used to treat the MDA-MB-231 cells. 24 hours later, the cells were harvested and lysed, the luciferase activity was determined by Dual-Luciferase Assay Kit (Promega).
Migration and invasion assay
Ishikawa cells were seeded in the upper chamber of 24 transwell inserts with 8-um pores coated with (for invasion assay) or without Matrigel (for migration assay). After indicated treatment, the cells were fixed and stained with crystal violet.
Apoptosis assay
The cells underwent various treatment were harvested and fixed overnight with cold 70% ethanol. After spinning down, the cell pellet was re-suspended in PBS and incubated with Annexin V. The apoptosis of the cells was analyzed by using flow cytometry.
Statistical analysis
SPSS 19.0 was used for analyzing the cellular growth, changes of western blot bands and apoptosis rate. P < 0.05 was considered significant difference when compared with control group.