Phosphate buffered saline (pbs)

Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium, trypsin (with EDTA), antibiotics (50 U/mL penicillin; 50 μg/mL streptomycin), fetal bovine serum (FBS), and phosphate-buffered saline (PBS) were purchased from HyClone Laboratories Inc. (Logan, UT, USA). PA, β-hydroxybutyrate, N-acetylcysteine (NAC, an antioxidant), and pyrrolidine dithiocarbamate (PDTC, an NF-κB inhibitor) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). p-IκBα, IκBα, NF-κB p65, and β-actin antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA), and histone H3 antibody was purchased from Abcam (Cambridge, MA, USA). Cell culture flasks were purchased from Thermo Fisher Scientific (Waltham, MA, USA), and six-well plates, 96-well plates, and filters were purchased from Nalge Nunc (Rochester, NY, USA).

Coronary artery SMCs grown on glass coverslips were washed in phosphate-buffered saline (PBS; Hyclone Laboratories, Inc.) and fixed in 4% paraformaldehyde (10 min at room temperature; Beyotime Institute of Biotechnology). Non-specific binding sites were blocked in 1% BSA (Hyclone Laboratories, Inc.) for 2 h. Cells were then incubated with a rabbit polyclonal anti-RAGE antibody (1:100 in blocking buffer) or a rabbit polyclonal anti-NF-κB p65 antibody (1:100 in blocking buffer). Following washing, cells were exposed to a goat anti-rabbit IgG-FITC (1:200) for 1 h at room temperature. Cells were mounted in 80% glycerol (Beyotime Institute of Biotechnology) and observed under a confocal microscope (LSM510; Zeiss, Oberkochen, Germany).

The in vitro circulation system mainly included a peristaltic pump (P-230, Harvard Instruments, Holliston, MA, USA), a silicone microtube (diameter 0.51 mm, length 1.5 m), and a syringe used as a reservoir for the cell solution, which could simulate fluid shear stress in the blood circulation by producing pulsating flow. In accordance with Poiseuille’s law, the wall shear stress τ (dyne/cm²) in the tubing was calculated by τ = 4µQ/(πR³), where Q is the flow rate (from 0.001 to 230 mL/min) and µ is the liquid dynamic viscosity (0.01 dyne/cm² for cell culture media), R is the radius of the tube (0.255 mm). The entire system was sterilized with 75% ethanol and then rinsed with 4 mL of phosphate-buffered saline (HyClone Laboratories, South Logan, UT, USA) before the experiment. To avoid the attachment of suspended tumor cells to the tubes and syringes, the system was treated with 4 mL of 1% bovine serum albumin (VWR Life Science, Radnor, PA, USA). In the process of the experiment, 2 mL of cell suspension (2 × 10⁵ cells/mL) was added into the circulation system and subjected to different magnitudes of shear stress for different durations in the cell culture incubator at 37 °C and 5% CO₂.

Anthraquinone (9,10-Anthraquinone), anthrarufin (1,5-dihydroxyAnthraquinone), chrysazin (1,8-dihydroxy-9,10-Anthraquinone), purpurin (1,2,4-trihydroxyAnthraquinone), 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azinobis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS), 3-tert-butyl-4-hydroxyanisole (BHA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), lipopolysaccharide (LPS, Escherichia coli 0111:B4), and other analytical grade reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA). 2′,7′-Dichlorofluorescein diacetate (DCF-DA) was purchased from Calbiochem (San Diego, CA, USA). Dulbecco’s modified Eagle’s medium, phosphate-buffered saline, fetal bovine serum, and other miscellaneous cell culture reagents were purchased from Hyclone Laboratories (Logan, UT, USA).

Anticancer agents interfere with various cell cycle phases. The cancer cell cycle may help elucidate the DNA-binding copper(II) complex mechanisms. HepG2 cells were inoculated onto six-well plates at 1 × 10⁵/well at 37 °C overnight under a 5% CO₂/95% O₂ conditions. The cells were left untreated or subjected to complex 1 or complex 2 for 24 h, trypsinized, rinsed with cold PBS (phosphate-buffered saline, Hyclone Laboratories Inc., Logan, UT, USA), fixed using 70% (v/v) ethanol, and kept at 4 °C. Afterwards, the pellets were washed twice with 1.0 mL PBS. Then, 10.0 mL of 20 μg/mL RNase (Sigma-Aldrich Corp., Shanghai, China) and 10 μL of 50 μg/mL PI (propidium iodide, Sigma-Aldrich Corp., Shanghai, China) were introduced, and the cell suspensions allowed to grow for 0.5 h at 37 °C. The samples were then analyzed with a Beckman CyAn-ADP flow cytometry platform (Beckman Coulter, Brea, CA, USA). Ten thousand cells were analyzed per sample. A similar procedure was used to assay the HepG2 cell cycles after 48 h treatment with complex 1 or complex 2.

High glucose Dublecco’s modified eagle medium (DMEM), L-glutamine 200 mM (100x), penicillin/streptomycin solution and phosphate-buffered saline (PBS) were purchased from Hyclone Laboratories (South Logan, UT). Fetal bovine serum (FBS) premium select was purchased from Atlanta Biologicals (Lawrenceville, GA). 96-well tissue culture plates were from Corning Inc. (Corning, NY).