Azoxymethane aom

Thirty-six BALB/c female mice (6–8 weeks old) were purchased for this animal study (Beijing Vital River Laboratory Animal Technology Co., Ltd.). The study involving the experimental animals was approved by the Institutional Animal Committee of Wenzhou Medical University. The mice were divided into three groups: control group, Fn + NaCl (Beyotime Institute of Biotechnology) group, and Fn + azoxymethane (AOM; 12 mg kg^–1; Sigma-Aldrich, United States) group, with 12, 10, and 14 mice in each group, respectively. Each mouse weighed about 20 g. After 1 week of acclimatization, AOM/0.9% NaCl was injected intraperitoneally once. One week later, the mice were given the Fn/0.9% NaCl by oral gavage, once a day, for 5 consecutive days. One week after stopping the intervention, mice were given the intraperitoneal injection of AOM/0.9% NaCl. One week after the second AOM/0.9% NaCl injection, Fn was given via another oral gavage for 5 days. Lastly, the mice were given water and normal food until the end of the experiment (81st day) (Supplementary Figure 1). The concentration of the intraperitoneal AOM injection was 10 mg/kg. The amount of Fn used was 1 × 10⁸ per mouse (OD_{600 nm} = 0.1) with 200 μL 0.9% NaCl.

Azoxymethane (AOM) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dextran sodium sulfate (DSS) was purchased from MP Biomedicals (Santa Ana, CA, USA). Antibodies of anti-P53, anti-gamma H2AX (phospho S139), anti-OXoguanine8, and anti-PCNA were purchased from Abcam (Cambridge, UK). Goat anti-mouse IgG (H+L) and goat anti-rabbit IgG (H+L) were purchased from Jackson Immuno-Research (West Grove, PA, USA). The Beta actin antibody was obtained from ZSGB-BIO (Beijing, China). In situ cell death detection kits, POD, were obtained from Roche Ltd. (Laval, QC, Canada). Phosphate buffer saline was obtained from Amresco (Solon, OH, USA). Liquid paraffin and neutral formalin were purchased from Beijing Chemical Works (Beijing, China).

Azoxymethane (AOM) (Sigma-Aldrich, Oslo, Norway) was diluted in 0.9% NaCl. All pups were subcutaneously (s.c) injected with 8 mg/AOM/ kg body weight first at age 7 ±1 days and secondly at 14 ±1 days, always keeping seven days between the two injections. All injections were made in the morning (between 8 and 11 a.m) to avoid possible differences in AOM metabolism.

Berberine was obtained from Sangon Biotech Co., Ltd. (Shanghai, China). Azoxymethane (AOM) was purchased from Sigma Aldrich (St. Louis, MO, USA). Dextran sulfate sodium salt (DSS) was provided by Dalian Meilun Biotech Co., Ltd. (Dalian, China). The AIN-93M rodent diet was purchased from Trophic Animal Feed High-tech Co., Ltd. (Nantong, China). All antibodies used in this study, were obtained from Zhengneng Biotechnology Co., Ltd. (Chengdu, China).

SHH inhibitors including cerulenin, cyclopamine, and itraconazole were purchased from Sigma-Aldrich (St. Louis, MO). Recombinant human TNF-α (rhTNF-α) was purchased from R & D Systems (Minneapolis, MN). Primary antibodies for western blotting were purchased as follows: ERK, pERK, NF-kB p50, FAS, FADD, XIAP, Survivin, and β-actin from Santa Cruz Biotechnology (Santa Cruz, CA); Cox-2 was from Thermo Scientific (Fremont, CA); other antibodies from Cell signaling technology (Danvers, MA). Horseradish peroxidase-conjugated anti-rat/rabbit/mouse/goat IgG was purchased Santa Cruz Biotechnology (Santa Cruz, CA). Primers for RT-PCR were synthesized by Bioneer (Seoul, Korea). Reverse transcriptase was from Promega (Madison, WI). Azoxymethane (AOM) was purchased from Sigma-Aldrich (St. Louis, MO) and dissolved in distilled water. Dextran sulfate sodium salt (DSS) was purchased from MP biomedicals (Morgan Irvine, CA). All other materials were obtained in the highest available grade.