Pcsk9

The proteins were prepared by homogenizing HepG2 cells in RIPA buffer ((1% v/v Triton X-100, 0.1% w/v sodium dodecyl sulfate, 0.5% w/v sodium deoxycholate, 10 mM sodium phosphate, pH 7.5, 5 mM EDTA, 5 mM EGTA, 100 mM sodium chloride, 1 mM PMSF, 20 M leupeptin, 20 mM methionine, and 1 mM cysteine)) containing a protease inhibitor cocktail on ice. The proteins were resolved on a 5% stacking, 8% running gel and transferred onto a nitrocellulose membrane. The membranes were blocked overnight with 5% nonfat dry milk in PBS with 0.1% Tween 20 and probed with primary antibodies overnight at 4 °C, followed by incubation with secondary antibodies for 90 min at room temperature. The membrane-bound antibodies were visualized using horseradish peroxidase-conjugated secondary antibodies (1:12,000) and the Odyssey Infrared Imaging System (Li-Cor Bioscience, Bad Homburg, Germany). Normalization was performed by blotting the same samples with an antibody against GAPDH. The primary antibodies were: PCSK9 (Abcam), LDLR (Abcam) and HNF1α (Abcam), each used at a dilution of 1:1000.

Cells and tissues were homogenized in a buffer containing 50 mM Tris HCl, 150 mM NaCl, 5 mM EDTA, 0.1% SDS, 1% sodium deoxycholate and protease and phosphatase inhibitor cocktails (Sigma) to isolate whole cell lysates. Isolation of the membrane fraction was performed with the Mem-PER^TM Plus Membrane Protein Extraction Kit (Thermo Fisher Scientific). After sonication and centrifugation, protein concentration was measured with the Pierce^TM BCA Protein Assay Kit (Thermo Fisher Scientific). 50 μg of protein were loaded on a SDS-PAGE gel and transferred to a nitrocellulose membrane. Following blocking, filters were incubated with antibodies directed against, phospho-AMPK (Thr172), alpha-AMPK, phospho-Akt (Thr308), total-Akt, phospho-AS160 (Thr642), total-AS160 (all Cell Signaling Technology), PCSK9 (Abcam), ND1 (Abcam), Cox I (Thermo Fisher Scientific), Na-K-ATPase and GAPDH (both Abcam). In order to rule out unspecific binding, the PCSK9 antibody was tested in tissue from PCSK9 knockout mice (Supplementary Figure 3C). After incubation with peroxidase-conjugated secondary antibody, blots were subjected to the enhanced chemiluminescent detection method with the Fusion FX7 imaging system (Peqlab).

The HepG2 human hepatocellular liver cell line was obtained from the Korea Research Institute of Bioscience and Biotechnology (South Korea) and grown in Eagle’s Minimum Essential Medium (EMEM) containing 10% fetal bovine serum and 100 U/mL penicillin/streptomycin sulfate. Cells were incubated in a humidified 5% CO₂ atmosphere at 37 °C. EMEM, penicillin, and streptomycin were purchased from Hyclone (Logan, UT, USA). Bovine serum albumin was purchased from Sigma-Aldrich (St. Louis, MO, USA). DiI-LDL was purchased from invitrogen (Invitrogen, Carlsbad, CA). Antibodies against LSS, SREBP1, SREBP2, PCSK9, HMGCR, LDL-R, SQLE and β-actin were purchased from Abcam, Inc. (Cambridge, MA, USA). PCSK9, LDL-R, HMGCR, SREBP1, SREBP2, HNF1A, BIRC2, ANXA2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) oligonucleotide primers were purchased from Bioneer Corp. (Daejeon, South Korea). Sauchinone was isolated from the chloroform fraction of Saururus chinensis, as previously described, and confirmed by a spectroscopic analysis^{32 (link)}. The purity of sauchinone was determined to be over 95% by high performance liquid chromatography using an ultraviolet detector.

The tissues of the carotid arteries and liver were homogenized in lysis buffer (20 mM Tris-HCl, 0.5% NP-40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 2 mM DTT, 0.5 mM phenylmethylsulfonylfluoride, 2 mM b-glycerophosphate, 1 mM sodium orthovanadate, 1 ug/mL leupeptin and pH 7.5, Sigma, USA), and then centrifuged for 30 min at 14,000 rpm at 4 °C so as to remove the supernatant and quantify the proteins by using a BCA assay kit (Bio-rad, Rockford, IL, USA). In addition, 50 ug of proteins were subjected to electrophoresis on a 9% SDS-PAGE and then transferred to a polyvinylidene difluoride (PVDF) membrane. The nonspecific reaction of the membrane was removed by blocking it for one h at room temperature in 5% nonfat dry milk in Tris buffered saline Tween-20 (TBST). PCSK9 (abcam, San Francisco, CA, USA), LDLr (Abcam, San Francisco, CA, USA), LOX-1 (Abcam, San Francisco, CA, USA), VCAM-1 (Cell signaling, USA) and beta-actin (Sigma, St. Louis, MO, USA) were incubated for 18 h at 4 °C in TBST with 5% nonfat dry milk. Horseradish peroxidase (HRP)-conjugated rabbit-antimouse IgG (Calbiochem, San Diego, CA, USA) was used for secondary antibodies. Blots were developed for visualization by using an enhanced chemiluminescence detection kit (Pierce Biotechnology, Rockford, IL, USA), and the Image Quant software (Molecular dynamics, Sunnyvale, CA, USA) was used to quantify the expression.

The numerous levels of protein expressions were measured by Western blot method. Anti‐Bax, Bcl‐2, Caspase 3, Cleaved‐ Caspase 3, anti‐cytochrome c (Cyt c) (Cell Signaling Technology, Danvers, MA, USA), PCSK9 (1:1000 dilution, Abcam, Cambridge, UK), LDLR (1:200 dilution, Abcam, Cambridge, UK) and anti‐actin (Sigma‐Aldrich, St. Louis, MO, USA) were used. In addition, the expressions of PCSK9, LDLR, CPT1 and complex I–V in oxidative phosphorylation (OXPHOS) in the liver were determined using the same methods used in cardiac tissues. The detection of horseradish peroxidase merged with antirabbit IgG was used to detect the bound antibody, and membranes were exposed to enhanced chemiluminescence (ECL).²⁹

The following antibodies were used: V5 was used to visualize GFP-Apex and S1R-Apex (ThermoFisher; 1:10,000 for western, 1:1,000 for immunofluorescence); Bip (Cell Signaling; 1:1,000 for western); PDI (Cell Signaling; 1:1,000 for western); Calnexin (Cell Signaling; 1:1,000 for western); Sec61alpha (Santa Cruz; 1:100 for western); Sigma1 Receptor (1:500 for western) (Ola et al., 2002 (link)); Lman1 (Abcam; 1:1,000 for western); PCSK9 (Abcam; 1:3,000 for western, 1:300 for immunofluorescence); LDLR (Novus biologicals; 1:1,000 for western, 1:100 for immunofluorescence); Thrombospondin (Abcam; 1:250 for western); Gapdh (Santa Cruz; 1:2000 for western).