Antibodies specific to the following proteins were obtained from Abcam (UK): LDLR (Abcam, #ab52818), LXRα (Abcam, #ab176323) and Idol (Abcam, #ab74562). GAPDH (#2118S, 1:1000) was obtained from Cell Signaling Technology (USA). EGCG (Sigma, #E4143) was obtained from Sigma (USA). Dil-LDL (Yiyuan biotechnology, # YB-0011) was purchased from Yiyuan biotechnology (China).

The human hepatoma cell line HepG2 was obtained from American Type Culture Collection and cultured in minimum essential eagle medium supplemented with 10% fetal bovine serum (FBS). All cells were maintained in a 5% CO2 humidified atmosphere at 37 ℃.

The RNA was isolated with Trizol (Invitrogen Life Technologies, California, USA) and reverse-transcribed to cDNA. The mRNA expression of LDLR, LXRα and Idol was determined by real-time PCR using specific primers, which were obtained from NCBI website (https://www.ncbi.nlm.nih.gov/). The SYBR Green real-time quantitative PCR assays were performed on a Lightcycler 480 II instrument (Roche Applied Science), and the melt curve was done. The quantitative analysis of mRNA expression was performed via 2−ΔΔCt method. The primer sequences are listed in Table 1.

At each time point, cells were harvested, washed with PBS, and homogenized with lysis buffer (50 mM Tris–HCl, pH 8.0, 5 mM EDTA, 150 mM NaCl, 0.5% NP-40, 0.5 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride), followed by shaking for 30 min at 4 ℃. Lysates were then centrifuged at 12,000 g for 10 min and supernatant was collected and quantified. Equal amounts of protein extract (20 μg or as indicated) were electrophoresed on a sodium dodecyl sulfate (SDS) polyacrylamide gel and transferred to a PVDF membrane (Millipore). The membrane was blocked with Tris-buffered Solution (20 mM Tris–HCl, 150 mM NaCl, and 0.1% Tween-20) containing 5% BSA for 1 h and probed with antibodies specific for LDLR (1:3000), LXRα (1:1000), Idol (1:1000) and GAPDH (1:10,000), respectively, at 4℃ overnight, followed by incubation with the corresponding secondary antibodies (Proteintech, Chicago, USA). The bands were visualized with enhanced chemiluminescence (ECL) on a Fuji LAS4000 (GE Healthcare).

HepG2 cells were incubated in serum-deficient medium for 24 h, then 25-OHC was added to the serum-containing medium for an additional 30 min, followed by the addition of EGCG of different concentrations (10, 25, 50 μM).To measure LDL uptake, Dil-LDL (20 μg/mL) (with red fluorescence) was added, followed by incubation at 37℃ for 4.5 h. The cells were washed with phosphate-buffered saline (PBS) three times. The LDLR activity of the HepG2 cells was determined by detecting the red fluorescence of the cells via a fluorescence microscope. The average fluorescence intensity was calculated by ImageJ software for quantitative analysis of LDL-uptake.

The continuous variables were presented as mean ± SEM. Student’s t-test was used to compare the difference between the two groups, and one-way ANOVA followed by Dunnett’s Multiple Comparison Test was used to compare the difference for more than two groups. P < 0.05 was considered statistically significant.