THP-1 human monocytic cell line was purchased from the Shanghai Cell Institute of the Chinese Academy of Sciences (Shanghai, China). THP-1 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, penicillin (100 U/ml) and streptomycin (100 µg/ml) at 37°C in 5% CO₂. To differentiate into macrophages, THP-1 monocytes were exposed to 160 nM phorbol-12-myristate-13-acetate (Sigma-Aldrich, St. Louis, MO, USA) for 72 h. The differentiated macrophages were washed three times with phosphate-buffered saline (PBS) and incubated in fresh serum-free medium containing 50 µg/ml oxidized LDL (ox-LDL; Yiyuan Biotechnology Co., Ltd., Guangzhou, China) for 48 h.

In order to investigate the effect of Trib1 overexpression in THP-1 macrophages, the cells were transfected with pCMV6-Entry-Trib1 (Trib1 overexpressing group) or pCMV6-Entry vector (empty vector group) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's instructions. The pCMV6-Entry-Trib1 and empty vector were purchased from OriGene Technologies, Inc. (Rockville, MD, USA). Non-transfected THP-1 macrophages were used as a control. For small interfering RNA (siRNA)-mediated knockdown experiments, THP-1 macrophages were co-transfected with pCMV6-Entry-Trib1 and LXRα siRNA or PPARγ siRNA (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). After 24 h of transfection, cells were incubated with ox-LDL (50 µg/ml) in serum-free medium for an additional 24 h before conducting the expression or functional studies.

Non-transfected or transfected THP-1 macrophages were cultured in chamber slides in serum-free medium. After 24-h incubation, cells were washed three times in PBS and fixed with 5% formalin solution for 30 min. Next, the cells were stained with oil red O (Sigma-Aldrich) for 30 min, and counterstained with hematoxylin for 5 min. Finally, cells were analyzed by light microscopy (Axio Imager 2; Zeiss, Oberkochen, Germany). Five selected high-power fields at a magnification of ×400 were randomly selected for examination. Semi-quantitative analysis of oil red O positive staining was conducted by the ImageJ software (version 1.48, National Institutes of Health, Bethesda, MD, USA).

In order to investigate the cholesterol efflux, non-transfected or transfected THP-1 macrophages (5×10⁵ cells/well) were seeded into 12-well plates, labeled with 0.5 µCi/ml [³H]-cholesterol (PerkinElmer, Waltham, MA, USA) in media containing 0.2% bovine serum albumin for 24 h and then washed with fresh media. Then, cells were washed with PBS and incubated in the presence of 10 µg/ml apolipoprotein A-I (apoA-I; EMD Millipore, Billerica, MA, USA) for 18 h. Medium and cell-associated [³H] cholesterol were examined through liquid scintillation counting. The percentage of cholesterol efflux was calculated with the following equation: [Total media radioactivity/(total cellular radioactivity + total media radioactivity)] × 100%.

Total RNA was isolated from THP-1 macrophages with TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Total RNA concentration was measured by spectrophotometry with a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Inc.). cDNA synthesis was performed using the PrimeScript RT reagent Kit according to the manufacturer's instructions (catalog no. RR037A; Takara, Shiga, Japan). qPCR analysis was then conducted using a Light Cycler-FastStart DNA Master SYBR-Green I kit (Roche Molecular Biochemicals, Manheim, Germany). The sequences of the primers used in the current study are shown in Table I. Data were analyzed with the 2^−ΔΔCq relative quantification method (11). Values are presented as fold change relative to control cells.

After the indicated treatment, cells were washed with ice-cold PBS and harvested in lysis buffer [30 mM HEPES, pH 7.6, 30 mM NaCl, 1% Nonidet P-40 (vol/vol), 10% glycerol (vol/vol), 50 mM NaF and 10 mM Na pyrophosphate] supplemented with 5 mM Na orthovanadate and protease inhibitors (Roche Diagnostics, Indianapolis, IN, USA). Cell lysates were collected by centrifugation at 14,000 × g for 5 min at 4°C, and the protein concentration in total lysates was analyzed by the BCA protein assay kit (Pierce; Thermo Fisher Scientific, Inc.). Next, 20 µg total protein were subjected to 10% SDS-PAGE and transferred onto a nitrocellulose membrane (Whatman; Sigma-Aldrich). Membranes were blocked overnight with 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h at room temperature. The membranes were then probed overnight at 4°C with the following primary antibodies: Anti-TRIB1 (ab137717; 1:200), anti-ABCA1 (ab18180; 1:200), anti-ABCG1 (ab155918; 1:200), anti-LXRα (ab82774; 1:200), anti-PPARγ (ab24509; 1:200) and anti-β-actin (ab97379; 1:500; all from Abcam, Cambridge, MA, USA). Subsequently, samples were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (ab97265; 1:2,000 dilution) or goat anti-rabbit IgG (ab97200; 1:2,000 dilution; both from Abcam) for 1 h at room temperature. Final detection was performed using an ECL chemiluminescence system (Bio-Rad Laboratories, Inc., Hercules, CA, USA), and quantitative results were obtained using Quantity One software (version 4.4.0; Bio-Rad Laboratories, Inc.).

All data are expressed as the mean ± standard deviation. Results were analyzed by one-way analysis of variance, followed by the Tukey's post hoc test, using SPSS version 11.0 software (SPSS, Inc., Chicago, IL, USA). P-values of <0.05 were considered to indicate differences that were statistically significant.

Transfection of THP-1 macrophages with pCMV6-Entry-Trib1 resulted in significant overexpression of Trib1 (increased by ~8.6-fold), as determined by western blot analysis (P<0.05 vs. vector-transfected cells; Fig. 1A). Subsequently, in order to explore the intracellular lipid deposition in response to Trib1 overexpression in THP-1 macrophages, the intracellular cholesterol levels were analyzed. Forced expression of Trib1 significantly inhibited lipid accumulation as evidenced by oil red O staining (P<0.05; Fig. 1B and C). Furthermore, exogenous expression of Trib1 significantly enhanced the apoA-I-mediated cholesterol efflux in ox-LDL-stimulated THP-1 cells (P<0.05; Fig. 1D). These results revealed that Trib1 inhibited the intracellular lipid deposition due to increased apoA-I-mediated cholesterol efflux in THP-1 macrophages.

RT-qPCR analysis demonstrated that overexpression of Trib1 significantly increased the ABCA1, ABCG1, LXRα and PPARγ mRNA expression levels in ox-LDL-stimulated THP-1 macrophages (P<0.05; Fig. 2A). Similarly, western blot analysis indicated that Trib1 overexpression led to a significant (1.5–2-fold) increase in the levels of ABCA1, ABCG1, LXRα, and PPARγ protein (P<0.05; Fig. 2B).

Silencing of LXRα by siRNA transfection significantly decreased Trib1-induced cholesterol efflux in THP-1 macrophages transfected with pCMV6-Entry-Trib1, when compared with the cholesterol efflux in THP-1 macrophages-transfected with pCMV6-Entry-Trib1 and a control siRNA (P<0.05; Fig. 3A). Furthermore, knockdown of PPARγ by siRNA transfection also significantly attenuated the Trib1-induced cholesterol efflux compared with transfection with a control siRNA (P<0.05; Fig. 3B).