Dulbecco modified eagle medium (dmem)

HCC cell lines, Hep3B and HepG2, were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), and cultured in DMEM (HyClone, Logan, UT) medium supplemented with 10% fetal bovine serum (FBS, GibcoBRL; Grand Island, NY, USA) at 37 °C with 5% CO₂. MDA19 (Cat# HY-15451, MedChemExpress, USA) was dissolved in DMSO and then diluted with DMEM to a specific concentration to incubate with HCC cells. siRNA targeting CB₂ and a negative control siRNA (siNC) were synthesized by Guangzhou RiboBio Co., Ltd. and transfected into HCC cells by using Lipofectamine2000 liposomes (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The sequences of siRNAs were as follows:
CB₂ siRNA1: 5′-CCAGGTCAAGAAGGCCTTT-3′;
CB₂ siRNA2: 5′- GCTTGGATTCCAACCCTAT-3′;
CB₂ siRNA3: 5′-CCTGGCCAGTGTGGTCTTT-3′;
siNC: 5′-UUCUCCGAACGUGUCACGUTT-3.

The ATC cell line C643 was purchased from Procell Life Science & Technology Co., Ltd., and OCUT-2C cells were purchased from iCell Bioscience Inc., Shanghai. C643 cells were cultured in RPMI 1640 (Gibco), and OCUT-2C cells were cultured in DMEM (Gibco), medium with 10% fetal bovine serum (FBS), and 1% penicillinstreptomycin solution (100 μg/ml, Leagene Biotech Co., Ltd.). The cells were incubated at 37 °C in a humidified 5% CO₂ atmosphere. Ibuprofen (purity ≥ 99.93%) was purchased from MedChemExpress and was directly dissolved in RPMI 1640 or DMEM. Ibuprofen was dissolved the day before, and the solution was put in a water bath at 37 ℃ to aid in the dissolving process.

Isotalatizidine was extracted and purified by the Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, and the purity was validated as > 95% using high-performance liquid chromatography. For the experiments, it was dissolved in dimethyl sulfoxide (DMSO) and subsequently diluted in sterile saline (0.9%). SB203580 and U0126-EtOH were all purchased from TargetMol (Shanghai, China). KG-501 was purchased from MedChemExpress (Shanghai, China) and dissolved in DMSO, and diluted with DMEM, DMEM/F-12, or saline before use. The cell culture reagents were purchased from the Invitrogen Corporation (Thermo Fisher Scientific, Carlsbad, CA, USA). Anti-dynorphin A antibody was purchased from Abcam (Cambridge, UK), and the Alexa 546-conjugated goat anti-rabbit and Alexa 488-conjugated goat anti-mouse secondary antibodies from the Life Technology (Thermo Fisher Scientific, Carlsbad, CA, USA). The remaining antibodies were purchased from the Cell Signaling Technology (Beverly, MA, USA). The goat serum was purchased from the Beyotime Biotechnology (Shanghai, China), and triton X-100 from the Sigma Aldrich.

The mouse photoreceptor-derived (661w) cell line was obtained from Professor Muayyad R. AI-Ubaidi of the University of Oklahoma. The cells were cultured in DMEM (Gibco) containing 10% foetal bovine serum (Biological Industries) and 1% antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin) (HyClone) in a humidified 5% CO₂ atmosphere at 37°C. The medium was replaced every 1 or 2 days. The cells were washed with PBS (Solarbio) before the experiments. For AGE treatment (Bioss, China, catalogue number: bs-1158P), AGEs were a white powder sourced from glycated BSA (purity: 98%). AGEs were dissolved at a concentration of 5 mg/ml in DMEM without foetal bovine serum and stored at -20°C. Metformin (Sigma) was dissolved at concentration of 50 mM in PBS and stored at -20°C. Compound C(MedChemExpress, USA) was dissolved at 1 mM in DMEM without foetal bovine serum and stored at 4°C.

Briefly, BMSCs were treated with DMEM (HyClone, United States ) containing 1, 2, 5, 10, 20, and 40 μM hydroxycholesterol (MedChemExpress, New Jersey, United States ); normal DMEM was used as a blank control. The cells were seeded at a density of 1 × 104 cells per well in the 96-well plates. After 6 h, the culture medium was replaced with the aforementioned hydroxycholesterol-containing medium. After incubation for 24 h, cell viability was detected with cell counting kit-8 (Dojindo, Japan), according to the manufacturer’s instructions, and the optical density (OD) was measured at 450 nm (OD450) to calculate the cell relative viability. Cell relative viability = (ODexperiment-ODblank)/(ODcontrol-ODblank) × 100%.