HASMCs were obtained from Lifeline Cell Technology (Beijing, China) and cultured according to the manufacturer’s instructions. The cells were cultured in Vasculife SMC cell culture medium, containing VascuLife basal medium and LifeFactors SMC (cat no. LS-1040; Lifeline Cell Technology) at 37°C in an atmosphere of 95% air and 5% CO₂ according to the manufacturer’s instructions. Cells were seeded in six-well plates and the culture medium was changed daily. Prior to each experimental treatment, cells were serum starved for 24 h under serum-free conditions. The concentration of human ox-LDL (24.5 nmoles of MDA/mg protein; Qingdao Haicon Biotechnology Co., Ltd., Qingdao, China) was primarily based on a previously published study demonstrating their effectiveness in vascular smooth muscle cells (21). According to these results, 100 μg/ml ox-LDL was used to stimulate HASMCs for different time periods, which were divided into 1, 2, 4, 6, 12 and 24 h subgroups. Subsequently, the strongest expression time point of PRR was selected and cells were treated with different concentrations of ox-LDL, which were divided into 25, 50, 100, 150, 200 and 300 μg/ml subgroups. At the end of each experiment, cells were harvested for the preparation of whole-cell lysates and total RNA extraction.

The expression of PRR in HASMCs was detected using immunofluorescence staining. HASMCs were seeded at a density of 5×10⁴ cells/ml into 24-well plates, cultivated and divided into groups. At the end of culture, the HASMCs were fixed with paraformaldehyde for 30 min. Cells were incubated with the primary antibody (polyclonal rabbit anti-human ATP6IP2 antibody; 1:700 dilution; ab64975; Abcam, Cambridge, MA, USA) at 4°C overnight. Following incubation with the fluorescein-labeled secondary antibody (fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin G; 1:200; sc-2012, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 37°C for 40 min, HASMCs were observed using fluorescence microscopy (DM4000 B LED; Leica, Mannheim, Germany) and images were immediately captured. The same process without the primary antibody and secondary antibody was used as a negative control. Green fluorescence of the cell membrane indicated positive expression of PRR.

Total RNA was extracted immediately from cultured cells using TRIzol reagent (Gibco-BRL, Dalian, China) according to the manufacturer’s instructions. Total RNA was reverse transcribed using the Prime Script RT reagent kit with gDNA Eraser (Perfect Real Time; Takara Bio Inc., Dalian, China). RT-qPCR was performed using SYBR Premix Ex Taq™ II (Takara Bio Inc.) with the Roche LightCycler^® 480 Sequence Detection System (Roche Diagnostics GmbH, Mannheim, Germany). Samples were run in triplicate in separate tubes to permit quantification of the target gene normalized to GAPDH, which was used for equal loading. Primer sequences are shown in Table I. The PCR amplification program was as follows: 95°C for 30 sec, 95°C for 5 sec and 60°C for 20 sec for 40 cycles.

Cells were lysed in 100 μl of lysis buffer (Nanjing KeyGen Biotech. Co., Ltd., Nanjing, China). Protein concentrations were measured using a protein assay kit (Nanjing KeyGen Biotech. Co., Ltd.). The protein (25 μg) obtained from each lysate was electrophoresed on a 15% SDS-polyacrylamide gel (90 mV, 30 min; 130 mV, 90 min) and transferred onto a nitrocellulose membrane (Merck Millipore, Darmstadt, Germany) using a Mini-PROTEAN 3 system (120 mV, 1.5 h; Bio-Rad, Hercules, CA, USA). The membrane was incubated overnight with polyclonal rabbit anti-human ATP6AP2 antibody (1:700 dilution; Abcam) and polyclonal rabbit anti-human β-actin (1:500 dilution; bs-0061R; Bioss, Beijing, China). The specific binding was detected using horseradish peroxidase-labeled goat anti-rabbit IgG (1:7,500; ZSGB-BIO, Beijing, China) and an enhanced chemiluminescence detection kit (Perkin-Elmer, Waltham, MA, USA). The bands were quantified using Image Pro-Plus 5.0 software (Media Cybernetics, Rockville, MD, USA).

Standard statistical methods from the SPSS statistical analysis system 16.0 (SPSS, Inc., Chicago, IL, USA) were used. Statistical comparisons were made using a two-way analysis of variance. Data are presented as the mean ± standard error of the mean. P<0.05 was considered to indicate a statistically significant difference. Duplicate wells were analyzed for each experiment and each experiment was performed independently at least three times.

Immunofluorescence techniques verified that PRR was expressed in HASMCs (Fig. 1), indicating that PRR was abundant in HASMCs and mainly present in the cell membrane and cytoplasm.

HASMCs were incubated with a final concentration of 100 μg/ml ox-LDL for 0, 1, 2, 4, 6, 12 and 24 h. It was found that the expression of PRR was significantly upregulated by ox-LDL in a time-dependent manner (Fig. 2). The mRNA and protein expression of PRR began to increase 2 h following incubation and reached a peak level at 6 h and then decreased moderately, however, maintaining a higher level of expression than the control group at 24 h.

Based on the above results, HASMCs were incubated with 0, 25, 50, 100, 150, 200 and 300 μg/ml ox-LDL for 6 h, respectively. Compared with the control group, the expression of PRR mRNA increased in the group treated with 25 μg/ml, reached a peak in the 50 μg/ml group, then decreased moderately in the remaining groups (Fig. 3A), revealing a concentration-dependent effect. The expression of PRR protein was similar to that of the mRNA, however, maintained significantly higher levels than the control group following decrease (Fig. 3B).