*2.14. Intracellular ROS Assay*

The fluorescent probe, dichlorofluorescin diacetate (DCFH-DA), purchased from Enzo Life Sciences Inc. (Farmingdale, NY, USA), was used to determine the effect of HLP on intracellular ROS generation by TNF-α stimulation. In brief, the confluent A7r5 cells in the 6-well plates at 10<sup>5</sup> cells/well were 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. After removing the treated cells from the wells, the cells were incubated with 2 μM of DCFH-DA at 37 ◦C for 30 min. The fluorescence intensity of intracellular ROS production was evaluated at an excitation and emission wavelength of 485 and 530 nm, respectively, using Muse™ Cell Analyzer (EMD Millipore Corporation, Merck Life Sciences, KGaA, Darmstadt, Germany). Values were expressed relative to the fluorescence signal of the control.

## *2.15. Evaluation of Atherosclerotic Lesions In Vivo*

New Zealand white male rabbits weighing between 1800 and 2200 g were randomly divided into five experimental groups as follows: Group I, normal control group (Purina Lab Diet 5031); group II, high-fat diet (HFD); group III, HFD with 0.5% HLP group (HFD + 0.5% HLP); group IV, HFD with 1% HLP group (HFD + 1% HLP); and group V, normal diet with 1% HLP group (cytotoxicity group of HLP). The rabbits in groups II, III, and IV were fed on a HFD containing 95.7% standard Purina Chow (Purina Mills Inc., Louis, MI, USA), 1.3% cholesterol, and 3% lard oil (Sigma-Aldrich, St Louis, MO, USA) for 25 weeks to induce the atherosclerotic process., In groups III, IV, and V, the rabbits were treated with oral feeding 0.5% or 1% HLP at the same time. The dose regimen for these groups was based on a previous study published by Chiu et al. [18]. For the care and use of laboratory animals, the use of all rabbits was reviewed and approved by Chung Shan Medical

University animal care committee according to the guidelines of the Institutional Animal Care and Use Committee (IACUC approval number: 893). After 25 weeks of supplementation, aortic arches from each rabbit were collected and then stained with hematoxylin and eosin (H & E) for the pathological analysis. Serum was also collected and stored at –80 ◦C until measurements of serum biochemical parameters and TNF-α using a cytoscreen immunoassay kit (BioSource International, Camarillo, CA, USA). For immunohistochemistry (IHC), commercial monoclonal anti-alpha smooth muscle actin (α-SMA, a marker of VSMC migration), obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA), and anti- PCNA (a marker of VSMC proliferation) were used for target detection in the paraffin-embedded tissues.
