Gsk3β

Total protein was extracted from the tumor tissue using RIPA protein extraction reagent containing phosphatase and protease inhibitors. The total protein concentration was measured using a BCA Protein Assay Kit. Then, the protein samples were mixed with a loading buffer (containing β-mercaptoethanol) and were heated at 100 ℃ for 10 min. Immunoblotting was performed using Bcl2-Associated X Protein (BAX) (Abcam, ab32503), B-cell lymphoma 2 (Bcl-2) (Abcam, 182858), SLC7A11 (Huabio, HA600097), GPX-4 (Bioss, 3884R), p53 (Proteintech, 60283-2-1g), GSK-3β (Huabio, ET1607-71), p-GSK-3β (Bioss, 2066R), Nrf-2 (Santa Cruz, 365949), cluster of differentiation 31 (CD31) (Huabio, ER31219), vascular endothelial growth factor-A (VEGFA) (Huabio, ET1604-28), MMP2 (Proteintech, 10373-2-AP), MMP9 (Proteintech, 10375-2-AP), Histone-H3 (Proteintech, 17168-1-AP), and β-actin (Proteintech, HRP-60008). Densitometry was performed using ImageJ software.

Madecassic acid (CAS no. 18449-41-7) with a purity of 98.5% was purchased from Jiangsu Yongjian Pharmaceutical Technology Co., Ltd (Taizhou, China). DSS (MW: 36–50 kDa) was purchased from MP Biomedicals Inc. (Irvine, CA, USA). Mouse IL-17 ELISA kit was purchased from Lianke Biotech Co., Ltd (Hangzhou, China). MPO activity assay kit was obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). FITC-anti-mouse CD3, PE-anti-mouse TCR γ/δ, PE-anti-mouse CD4, PE-anti-mouse CD8, and APC-anti-mouse IL-17A were purchased from eBioscience, Inc. (San Diego, CA, USA). Murine IL-1β and IL-23 were purchased from Sino Biological Inc. (Beijing, China). Mouse TCRγ/δ T Cell Isolation Kit was purchased from Miltenyi Biotec Inc. (Bergisch Gladbach, Germany). TRIzol reagent was purchased from SunShine Biotechnology Co., Ltd (Nanjing, China). PPARγ, phosphorylated (p)-PPARγ, PTEN, p-PTEN, PI3K, p-PI3K, Akt, p-Akt, GSK3β, p-GSK3β, mTOR, p-mTOR, NFATc1, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mAb were purchased from HuaBio Inc. (Hangzhou, China). Cyclosporin A, GW9662, MK-2206, and rapamycin were obtained from CSNpharm (Chicago, USA). Rosiglitazone, FK506, LY294002, and SC-97 were purchased from Selleck (Houston, USA).

The proteins were extracted and measured using the BCA Protein Assay Kit (Thermo Fisher, USA). The denatured proteins were electrophoretically separated and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, USA). After blocked with 5% BSA for 1 hour at room temperature, the membranes were immersed with the primary antibodies (Collagen II, 1 : 1000, Cell Signaling Technology, USA; LC3, 1 : 1000, Abcam, USA; p-mTOR, 1 : 1000, Cell Signaling Technology, USA; mTOR, 1 : 1000, Cell Signaling Technology, USA; GSK3β, 1 : 1000, Huabio, China; p-GSK3βser9, 1 : 1000, Huabio, China; β-catenin, 1 : 1000, Cell Signaling Technology, USA; GAPDH, 1 : 2000, Abcam, USA) overnight at 4°C, respectively. After washed with TBST (Beyotime Biotechnology, China), the membranes were incubated with IgG conjugated to horseradish peroxidase (1 : 2000; Santa Cruz Biotechnology, USA). The protein bands were visualized by using an ECL kit (Merck Millipore, USA). The relative expression level of specific protein was quantified by the optical density of the target band against that of GAPDH using a Bio-Rad image analyzer (Bio-Rad, USA).