Foetal bovine serum (fbs)

Cardiomyocytes were isolated from sham and CHF rats and cultured, following a previous procedure 15. Hearts were removed from anaesthetized rats, mounted on a Langendorff system, and retrogradely digested with calcium‐free Krebs–Henseleit buffer containing 1 mg/ml collagenase type 2 (Worthington Biochemical, Lakewood, NJ, USA), saturated with 95% O₂‐5% CO₂ at 37°C. When the heart became flaccid, the ventricles were minced, and digested further in a shaking water bath. Dissociated cells were then collected, brought back to calcium‐containing buffer, pre‐plated to remove fibroblasts, and cultured with DMEM (Sigma‐Aldrich Corp., St. Louis, MO, USA) supplemented with 10% foetal bovine serum (HyClone Laboratories, Logan, UT, USA) in a CO₂ incubator at 37°C. The cardiomyocytes were cultured for 24 hrs before downstream experiments, including TLR4 binding and function assay.
To determine the pro‐inflammatory function of TLR4, cultured cardiomyocytes were treated with the PAMP ligand lipopolysaccharide (LPS, Cat. L4524; Sigma‐Aldrich Corp.), or the DAMP ligand heat shock protein 60 (HSP60, low endotoxin, Cat. ADI‐ESP‐741; Enzo Life Sciences, Inc., Farmingdale, NY, USA). Toll‐like receptor 4 neutralizing antibody (anti‐TLR4) was added 15 min before the treatment of LPS or HSP60.

COS7 (obtained from RIKEN Cell Bank) and RBL-2H3 (obtained from American Type Culture Collection) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Nacalai Tesque) containing 10% foetal bovine serum (Hyclone Laboratories), 50 U/ml penicillin, and 50 μg/ml streptomycin (Thermo Fischer Scientific) at 37 °C and 5% CO₂. COS7 cells (4 × 10⁵ cells) were transfected with 0.5–2 μg per plasmid using Lipofectamine 2000 transfection reagent (Thermo Fischer Scientific), and RBL-2H3 cells (6 × 10⁶ cells) were electroporated in 600 μl DMEM with 25–50 μg per plasmid and/or with 200 nM siRNA at 250 V and 950 μF, using the Gene Pulser Xcell device (Bio-Rad). To concentrate the RBL-2H3 transfectant, 24 h after electroporation, G418/geneticin (InvivoGen) was added to the medium to the final concentration of 0.6 mg/ml and the culture was continued for additional 24 h. Thereafter, 48 hours after electroporation, the surviving cells were subjected to subsequent analyses.

NIH3T3 cells were cultured in high‐glucose Dulbecco's modified Eagle's medium containing 10% foetal bovine serum (HyClone Laboratories). Cells were changed to a fresh medium containing 5 μg/mL hexadimethrine bromide (polybrene) and infected with a lentivirus (multiplicity of infection, 50). Cells were then switched to a fresh culture medium. Puromycin (2 μg/mL) was added to the medium 48 hours after infection. Next, the cells were cultured in medium with 2 μg/mL puromycin and regularly changed medium. After several passages, the stable cells were used for subsequent studies.

Murine melanoma B16-F1 cell line (CRL-6323) was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in Dulbecco’s modified Eagle’s Medium (DMEM; Nacalai Tesque Inc., Kyoto, Japan) supplemented with 10% (v/v) foetal bovine serum (HyClone Laboratories, Logan, UT, USA) and 1% (v/v) penicillin G (100 U/mL) and streptomycin (100 µg/mL) (gibco, Thermo Scientific, Waltham, MA, USA). The cells were grown and maintained at 37 °C with 5% CO₂ and humidity. Monoclonal antibodies against phospho-p38, total p38, and cleaved caspase 3 were obtained from Cell Signaling Technology (Danvers, MA, USA). A monoclonal antibody against β-actin was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Phosphate buffered saline (PBS), 0.05% trypsin, and phenol red-free DMEM were purchased from Gibco (Thermo Scientific). Dimethyl sulfoxide (DMSO) was purchased from Kanto Chemical Co. (Tokyo, Japan). Methanol and absolute ethanol were purchased from Fisher Scientific (Thermo Scientific). MTT, αTP, synthetic melanin, l-3,4-dihydroxyphenylalanine (L-DOPA), bovine serum albumin (BSA), DCFDA, and JC-1 kit were obtained from Sigma-Aldrich (St. Louis, MO, USA). TRF with a purity of ≥95% was supplied by Davos Life Science Sdn Bhd (DavosLife E3, Petaling Jaya, Malaysia).

JHU020 and JHU006 human HNSCC cell lines were generated and genetically characterised at the Johns Hopkins University Head and Neck Biological Research Laboratories from human tumour explants. Both cell lines were propagated and carefully maintained in our laboratory. A large batch of both JHU020 and JHU006 cell lines were produced immediately prior to the beginning of the study. The majority of the cells were frozen while aliquots were sent for authentication using Short Tandem Repeat Analysis by BioSynthesis Inc. The cells were also tested for mycoplasma contamination. With the exception of those experiments that took place simultaneously, an aliquot of each cell line was thawed for every experiment. Long-term propagation did not occur. The cell lines were cultured in RPMI-1640 culture media (Gibco, Baltimore, MD), supplemented with 12% Foetal Bovine Serum (Hyclone Laboratories, South Logan, UT) and 1% Penicillin–Streptomycin solution (Corning, Manassas, VA). The cells were plated in 6-well plates at a density of approximately1.5 × 10⁵ cells/ml for JHU006 cells and 3 × 10⁵ cells/ml for JHU020 cells.