*2.1. Reagents*

Liensinine (Cas number: 2586-96-1) was purchased from Sigma Aldrich. 3-(4,5- Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lipopolysaccharide (LPS) were obtained from Sigma Aldrich (St Louis, MO, USA). PDGF-BB and TNFα were purchased from R & D systems (Minneapolis, MN, USA). All remaining common laboratory chemical reagents or solvents were purchased from Sigma-Aldrich, South Korea.

### *2.2. Cell Culture*

VSMC from human aorta obtained from ATCC, USA and RAW264.7 cells purchased from Korean cell line bank (Seoul, Korea) were cultured in complete cell culture media with Dulbecco's modified Eagle's medium (DMEM), 10% fetal bovine serum (FBS), and 1% antibiotics (penicillin + streptomycin) in a standard cell incubator with 5% CO2. The cells were incubated in media with 0.1% FBS for 24 h to allow them to synchronize at G0 phase for each assay. Liensinine was solubilized in dimethyl sulfoxide (DMSO) and diluted in a serum-free medium for treatment of cells. The final % of DMSO while treating cells were below 0.1%.

### *2.3. DPPH Assay and Thiobarbituric Acid Reactive Substance (TBARS) Assay for Lipid Peroxidation Assay*

The anti-oxidant activity of liensinine was evaluated using a DPPH free radical scavenging assay and measurement of serum lipid peroxidation was carried out using TBARS assay following the methods as described previously [11].

### *2.4. Proliferation Assay*

PDGF-BB was used as a proliferation inducer in VSMC and % proliferation was measured by MTT colorimetric assay as described previously [24]. VSMC were treated with liensinine 1 h before PDGF-BB and incubated for 24 h to allow proliferation. Freshly prepared MTT in phosphate buffer saline was added and incubated for an additional 4 h. The purple color formazan developed due to the reduction of MTT by viable VSMC were dissolved with DMSO. Then, a colorimetric reading was taken by measuring absorbance at 540 nm with a microplate reader. The anti-proliferative effect of liensinine was evaluated by comparison with the control group (treated with PDGF-BB) as 100%.

### *2.5. Gelatin Zymography*

Gelatin zymography was carried out to examine the enzymatic activity of MMP-9 as described previously [14]. Briefly, VSMC were seeded in 60 mm petri plates at the density of 1 × 10<sup>6</sup> cells. Liensinine was added at a predetermined concentration for 1 h and cells were treated with TNFα (100 ng/mL) for the next 24 h. The supernatant cell culture media was collected and 30 μg of protein equivalent was used for electrophoresis in 10% SDS-PAGE with 0.25% gelatin. Next, the gels were incubated in renaturating buffer (2.5% Triton X-100) for half-an-hour and incubated again in developing buffer at 37 ◦C for 16–24 h. In order to visualize the bands of MMP2 and MMP-9, gels were stained with 0.05% Coomassie Brilliant Blue followed by incubation in destaining buffer. Photographs of the gel were taken to observe the proteolysis of gelatin by MMP-2 and MMP-9.

### *2.6. Determination of IL-6 Release in TNF-α Stimulated VSMC*

VSMC was pretreated with 1–30 μM of liensinine for 1 h and further treated with TNFα for the next 24 h. The level of IL-6 released by cells was measured in culture supernatant using an ELISA kit of IL-6, according to manufacturer's protocol.

### *2.7. Cell Viability/Cytotoxicity Assay, NO Release and Immunoblot of iNOS, and COX-2 Protein Expression in RAW264.7 Cells*

RAW264.7 cell viability or cytotoxicity assay was carried out using the MTT colorimetric assay as described in Section 2.4 (without any stimulant). The NO levels in RAW264.7 cells were evaluated as mentioned previously [25]. Briefly, the cells were treated with 1–20 μM of liensinine for 1 h, then induced with LPS at 1 μg/mL for the next 24 h. The level of NO in culture supernatant was measured by mixing a 1:1 ratio (100 μL) of supernatant: Griess reagent. The colored product was measured calorimetrically by reading absorbance at 540 nm. For protein expression of iNOS and COX-2 in RAW 264.7 cells, immunoblotting was carried out [26].

### *2.8. Statistical Analysis*

Data analysis and graphs were prepared using SigmaPlot or Microsoft Excel. The data are represented as mean ± standard error mean. Multiple groups were compared using one-way analysis of variance (ANOVA) and Duncan's post-hoc test. *p*-values of < 0.05 were considered as statistically significant.
