*2.11. Bromodeoxyuridine (BrdU) Cell Proliferation Assay*

To identify the cells in S phase of cell cycle, the BrdU cell proliferation assay (Oncogene, Cambridge, MA, USA) was carried out according to the manufacturer's manual. In brief, A7r5 cells were seeded into a 96-well plate (4 × 10<sup>3</sup> cells/well) and grown in DMEM medium supplemented with 5% FBS overnight. The cells were rinsed once with serum-free medium, and then treated with TNFα (10 ng/mL) in the

presence or absence of various concentrations (0.2 and 0.5 mg/mL) of HLP in serum-free medium for 24 h. In most of the experiments, pulse labeling of synthesized DNA was used. For this, the BrdU label was added 1 h before the experimental end. The cells were fixed, denatured, and probed with anti-BrdU antibody. Absorbance was determined at dual wavelengths of 450 and 540 nm in a microplate reader system (Bio-Rad Labs., Hercules, CA, USA). Proliferative value (BrdU incorporation) was expressed as a percentage of absorbance of the treated cells to the absorbance of the non-treated control cells. The BrdU incorporation of the control group was set to 100%.

## *2.12. Cell Cycle Analysis by DNA Content*

The quantification of cell cycle distribution was examined using a FACScan laser flow cytometer (Becton Dickinson, San Jose, CA, USA). The VSMC was treated with TNF-α (10 ng/mL) in the absence or presence various concentrations (0.2 and 0.5 mg/mL) of HLP for 24 h; collected, rinsed with PBS twice; fixed in 70% ethanol at –20 ◦C overnight; and then stained with propidium iodide (PI) solution (20 μg/mL of PI, 20 μg/mL of RNase A, and 0.1% Triton X-100; all chemicals from Sigma-Aldrich, St Louis, MO, USA) for 20 min in the dark at room temperature. Each phase of cell cycle was presented as the cell number versus the DNA content as indicated by the intensity of fluorescence, and gated into subG1, G0/G1, S, and G2/M phases with CELLQuest Version 3.3 software (Becton Dickinson, San Jose, CA, USA).
