Fetal serum

For primary NK cells, single hepatic lymphocytes from 6- to 8-week-old C57BL/6 mice were labeled with PE-Cy7-conjugated anti-CD3, PE-conjugated anti-CD4, APC-conjugated anti-NK1.1 and APC-Cy7-conjugated anti-CD19 (BD PharMingen, United States). NK cells were sorted as cells that express NK1.1⁺CD3^- using a FACS Aria I flow cytometer (BD Biosciences, United States) and the purity was higher than >95%. After purification, NK cells were cultured in a 96 round bottom wells (Costar, Corning Incorporated, Corning, NY, United States) in complete RPMI 1640 medium supplemented with 10% (v/v) fetal serum (Gibco, United States), 200 mg/ml glutamine, 50 μg/ml gentamycin, 100 units/ml penicillin, 100 μg/ml streptomycin (Sangon Biotech, Shanghai, China) and interleukin-2 (IL-2, 10 U/ml, BD PharMingen, United States).

The human hepatocellular carcinoma cell lines (HepG2 and HUH-7), endothelial cells derived from hepatocellular carcinoma (ECDHCC-1), THLE-2, and L02 cells derived from healthy liver cells were all obtained from American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were cultured at 37°C with 5% CO₂ in Dulbecco's modified Eagle's medium (DMEM) high-glucose medium containing 10% fetal serum (Gibco-Life Technologies, Carlsbad, CA, USA) and 1% penicillin and streptomycin. To establish the sorafenib-resistant HCC cell lines, the half-maximal inhibitory concentration (IC₅₀) of HCC cells to sorafenib was initially determined by incubating cells with different concentrations of sorafenib in 96-well plates, and cell viability was measured 3 days later as described below. The cells were cultured in 6-well plates at 1 × 10⁴ cells/well and incubated with sorafenib at a concentration just below their respective IC₅₀. The concentration of sorafenib was slowly increased by 0.25 μM per week. After 6 to 7 months, sorafenib-resistant cell lines were obtained and continuously maintained in the presence of sorafenib. The resistance index (RI) was calculated as the ratio of IC₅₀ of sorafenib resistant cell line to IC₅₀ of corresponding control cell line.

HBV-DNA-Pol⁺ Huh7 cells or control cells were co-cultured with Jurkat cells at a ratio of 3:1 in complete RPMI medium 1640 basic (Gibco) supplemented with 10% fetal serum (Gibco) and penicillin–streptomycin for 24 h in 24-well plates with or without a well at 37 °C in a humidified incubator with 5% CO₂. Here, “with well” means that the two cells are cultured in a non-contact manner, i.e., indirectly, and “without well” means that the two cells are mixed together and cultured in a contact manner, i.e., directly. The “well” is an experimental tool that allows the spatial separation of cells. Then Jurkat cells were sorted out because of their suspension properties and stimulated by adding 20 μg/ml Concanamycin A (Con A) for 24 or 48 h for subsequent experiments. The supernatants were collected after 24 h and examined using a human or mouse IFN-γ/TNF-α ELISA kit (Solarbio, Beijing, China) according to the manufacturer’s instructions, and the results were analyzed using ELISACalc V0.1 software (Quant, Biotek, Germany).

The immortalized human NK cell line, NK92MI was obtained from the American Type Culture Collection (CRL-2408) and maintained in α-Minimum Essential Medium (Gibco) supplemented with 0.2 mM inositol (Sigma), 0.1 mM 2-mercaptoethanol (Gibco), 0.02 mM folic acid (Sigma), 12.5% fetal serum (Gibco), 12.5% horse serum (Gibco), and plated at 2-3 × 10⁵ viable cells/mL at 37°C and 5% CO₂ in T-75 culture flasks and passaged every 48h.

We obtained SW48, LoVo, HCT116, SW620, HT-29 and RKO cell lines from ATCC (Virginia, USA). The cell lines were cultured in high glucose Dulbecco’s Modified Eagle Medium (DMEM; Gibco, USA), supplemented with 10% fetal serum (Gibco, USA), 100 U/mL penicillin and 100 U/mL streptomycin.

SH-SY5Y cells were purchased from ATCC (Maryland, America). The cells in normal group were cultured in a normal culture medium containing DMEM solution (Gibco/Life Technologies Ltd, Paisley, Scotland) mixed with 10% fetal serum (Gibco/Life Technologies Ltd, Paisley, Scotland) and 7.5% horse serum (Gibco/Life Technologies Ltd, Paisley, Scotland), and incubated in a humidified incubator (Thermo CO₂ incubator, 311, USA) at 37? and 5% CO₂. The cells in OGD group were cultured in an ischemia-mimetic solution (mmol/L: 140 NaCl, 3.5 KCl, 0.43 KH2PO₄, 1.25 MgSO₄, 1.7 CaCl₂, 5 NaHCO₃, 20 HEPES, pH 7.2-7.4) and kept in a hypoxic incubator chamber (Billups- Rothenberg) filled with 95% N₂/5% CO₂ at 37? for 4, 8, or 12 hr. Ten percent of intralipid solution at a concentration of 50μM was used as a vehicle (Sigma, St Louis, MO). Rapamycin (RAP) (100 nm; Sigma, R0395) and 3-methyladenine (3-MA) (400 nmol; Sigma, M9281) were used as autophagy activator and inhibitor, respectively, and added to the medium 10 min before OGD.
After OGD for different time, the cells were transferred to normal culture medium and kept in an incubator with 5% CO₂ at 37?for 24 hr for reperfusion and all the following experiments.

CU (purity≥98%), 5-FU, RPMI 1640 medium, fetal serum, 0.25% trypsin, and 100 units/mL of penicillin-streptomycin were purchased from Thermofisher Company. MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5 diphenyl-2-H-tetrazolium bromide) were purchased from Luzhou Shuangjiang Chemical Co, Ltd (Sichuan, People's Republic of China). CU and 5-FU were dissolved in DMSO (dimethyl sulfoxide) and taken as 800 μmol/L solution with complete culture solution, in which a final concentration of DMSO was 0.1% (v/v), and then further diluted as needed in cell culture medium. The NF-κBp65 antibody, COX-2 antibody and β-actin were purchased from Santa Cruz Company.