Hoechst 33342 reagent

HT-29 and HCT-15 human colon carcinoma cells and MC-38 murine colon carcinoma cells were purchased from China Infrastructure of Cell Line Resources (China) and then cultured in DMEM and RPMI-160 medium, respectively, supplemented with 10% (v/v) fetal bovine serum (FBS) and 100 U/ml streptomycin/penicillin at 37 °C and 5% CO₂. Cell Counting Kit-8 (CCK-8) reagent was purchased from Dojindo (Japan). Hoechst 33342 reagent was purchased from Beyotime (China). An Annexin-V FITC apoptosis detection kit (#556547) and FITC BrdU cycle detection kit Part A (#559619) were purchased from BD Biosciences PharMingen (USA). Taxifolin was purchased from Aladdin (China). Antibodies against Bcl-2, Bax, caspase 9/p35/p10, poly (ADP-ribose) polymerase-1 (PARP1), cyclin D1, and beta-actin were purchased from Proteintech. Antibodies against cyclin A2, cyclin B1, p-Akt (S473), p-Akt (T308), pan-Akt, p-ERK (T202/Y204) and pan-ERK were purchased from CST. Antibody against CDK2 was purchased from Abcam. CR powder was provided by the Chinese Medicine Pharmacy of Tianjin Medical University Cancer Institute and Hospital. The concentration of the prepared CR solution was 0.05 g/ml. In addition, for the subsequent relevant biological experiments, the 0.05 g/ml solution was subjected to 0.22 μm filter membrane filtration and sterilization and dilution to the necessary concentrations.

After deparaffin, thin sections (5 μm) of the liver tissues were blocked with 1% bovine serum albumin, and then they were incubated with the primary antibodies overnight at 4°C. After three washes with PBS, sections on slides were incubated with fluorescence-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA) at room temperature for 2 hrs. Sections incubated with secondary antibodies alone were used as negative controls. For staining with cells, HSCs (50,000 cells/well) were seeded in 6-well plates and cultured in DMEM with 10% FBS for 24 hrs and then in low-serum medium (0.5%, v/v) for an additional 12 hrs. Hepatic stellate cells were then treated with various reagents as indicated in specific experiments for 24 hrs. Cells were incubated with the primary and secondary antibodies in succession similar to the above described protocol. Finally, hoechst 33342 reagent (Beyotime Institute of Biotechnology, Haimen, China) was used to stain the nucleus. Representative micrographs are shown.

Iron(III) acetylacetonate (Fe(acac)₃), oleic acid, oleylamine, cyclohexane, N-hydroxysuccinimide (NHS), D-cysteine hydrochloride monohydrate (Cys), and Cy5.5-NHS ester were purchased from Aladdin. Tetrahydrofuran (THF) and acetone was purchased from Sinopharm Chemical Reagent Co., LTD., Shanghai, China. The 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) was purchased from Energy Chemical. Polyethylene glycol (M_w ≈ 2000 Da) with a diphosphate group at one end and a maleimide group at the other end, denoted as DP-PEG-Mal, was provided by Suzhou Xinying Biomedical Technology Co., LTD., Suzhou, China. Boc-Cys(Trt)-OH was purchased from Shanghai Maclin Biochemical Technology Co., LTD., Shanghai, China. The 1-(2-Aminoethyl)-2-methyl-5-nitroimidazoledihydrochloride was purchased from Acros Organics. Triethylsilane was purchased from TCI. Dulbecco’s modified eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from HyClone. Hoechst 33342 reagent was purchased from Beyotime Biotech. HIF-1α mouse monoclonal antibody was purchased from Affinity.