Celastrol

Human colorectal adenocarcinoma cell lines HCT116 and HT-29 were obtained from Shanghai Institute of Cell Resource Center of Life Science (Shanghai, China). All cells were cultured in McCOY’s 5A medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone, USA), 100 mg/ml streptomycin and 100 U/ml penicillin at 37°C in humidified atmosphere with 5% CO₂.
Male C57BL/6 mice (n = 65, 6–8 weeks old) used for UC-CRC models and male BALB/c-nu mice (n = 36, 5 weeks old) used for colorectal tumor xenograft models were purchased from Vital River Laboratory Animal Technology Co. Ltd (Beijing, China). All animals were housed under controlled conditions (temperature 22 ± 1°C, humidity 40–60% and 12 h dark/light cycle) and free access to a standard laboratory diet and water for 2 weeks. All animal care and experimental procedures were carried out in accordance with the recommendation of the Animal Care Ethics and Use Committee of China Medical University and approved by this Committee.
Celastrol (≥98%) was purchased from Dalian Melone pharmaceutical Co., Ltd (Dalian, China). For in vitro studies, Celastrol was dissolved in dimethyl sulfoxide (DMSO; Sigma–Aldrich) at a stock concentration of 44 mM. For animal experiments, Celastrol was dissolved in DMSO at 20 mg/ml and then diluted with 0.9% saline to the final concentrations (1% DMSO) before administration.

Tamoxifen was purchased from Tocris (Minneapolis, MN). Celastrol was purchased from Shanghai Hotmed Sciences Co. Ltd. (Shanghai, China), with 98% purity or higher. Dimethyl sulfoxide (DMSO) was purchased from Sigma (St. Louis, USA). Tamoxifen and Celastrol were dissolved in DMSO (0.1%, v/v, final concentration), then sterilized by a 0.22 μm pore filter (Merck Millipore, Bedford, USA). Antibodies were purchased from the following companies: LC3I/II, P62, phosphor-Akt, and phosphor-mTOR from Cell Signaling Technology (Danvers, UK); β-actin, anti-mouse-IgG, and anti-rabbit-IgG from Santa Cruz Biotechnology (Santa Cruz, CA, USA); and Hoechst 33342 from Guangzhou Ribobio (Guangzhou, China). The Annexin V-FITC Apoptosis Detection Kit and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from KeyGEN Biology Co. (Nanjing, China). Alexa Fluor 488 goat anti-rabbit-IgG (A11008) was purchased from Invitrogen (Carlsbad, CA).

Celastrol (purity ≥98%) was purchased from Sigma (St. Louis, MO, USA) and dissolved in 10% dimethyl sulfoxide (Sigma). It was diluted with cell culture solution prior to treatment to ensure that dimethyl sulfoxide had no effect on the cells. Primers for monocyte chemoattractant protein (MCP)-1, caspase1, pdk4, and foxo1 were synthesized by Invitrogen (Carlsbad, CA, USA). Primary antibodies were purchased from either Abcam (Cambridge, UK) or Cell Signaling Technology (Danvers, MA, USA). Enzyme-linked immunosorbent assay (ELISA) kits for MCP-1, IL-10, catalase (CAT), and superoxide dismutase (SOD) were purchased from Immunoway (Plano, TX, USA), R&D Systems (Minneapolis, MN, USA), and Abcam.