The human acute monocytic leukemia cell line, THP-1, was purchased from the Shanghai Institute of Cell Resource Center of Life Science (Shanghai, China), and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) (GE Healthcare Life Sciences, Logan, UT, USA) and 100 U/ml penicillin-streptomycin (Life Technologies, Grand Island, NY, USA), as growth medium, in a humidified incubator at 37°C with 5% CO₂.

The pcDNA3.1-AMFR-C plasmid, encoding a 50kDa truncated form of AMFR (AMFR-C), was provided by Professor Le-xun Xue (16), and transfected into the THP-1 cells by electroporation using an Electro Cell Manipulator (BTX Harvard Apparatus, Holliston, MA, USA). Briefly, 1×10⁷ THP-1 cells were cultured in penicillin-streptomycin-free RPMI-1640 medium at 37°C for 24 h prior to transfection. The cells were harvested, washed twice with phosphate-buffered saline (PBS) and resuspended in 0.8 ml medium containing 20% FBS. The cell suspension was divided into two electro cell cups, to which 15 µg of the pcDNA3.1 or pcDNA3.1-AMFR-C plasmids were added. Following mixing for 5 min, a current of 200 V and 950 UF was applied at 37°C for 5–10 min, and the cell suspension was transfered into a 100 ml culture flask containing 10 ml growth medium. The cells were harvested 72 h after transfection.

Subsequently, the THP-1 cells were transiently transfected with AMFR siRNA (cat. no. sc-43809; Santa Cruz Biotechnology, Inc.) or scrambled siRNA (cat. no. sc-36869; Santa Cruz Biotechnology, Inc.), a non-target fluorescein isothiocyanate (FITC)-conjugated control, in small interfering (si)RNA transfection reagent (car. no. sc-29528; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), according to the manufacturer's instructions, as previously described (28,29). Transfection was detected using fluorescent microscopy (ECLIPSE TE2000-U; Nikon Corporation, Tokyo, Japan), and a transfection efficiency of 60–70% was achieved.

At 24, 48, 72 and 96 h post-transfection, total RNA was extracted from the THP-1 cells using an RNA Prep Pure Cell kit (Tiangen Biotech Co., Ltd., Beijing, China). The RNA purity was determined according to the absorbance at 260 and 280 nm (A260/280; ultraviolet spectrophotometry, U-2001; Sartorius Corporation, Göttingen, Germany) and the RNA was reverse transcribed into cDNA using a RevertAid™ First Strand cDNA Synthesis kit (MBI Fermentas, Inc., Burlington, ON, Canada), according to the manufacturer's instructions. The expression of AMFR was detected by qPCR using a SYBR-Green kit (Tiangen Biotech Co., Ltd.) with the following primers (Sangon Biotech, Shanghai, China): Forward 5′-GTCAGGGAAGAACATCAAGGAG-3′ and reverse 5′-GGGGAAACATCTCTTGAATCTG-3′. β-actin, used as an internal control, which was detected uising the following primers: Forward 5′-CCCATCTATGAGGGTTACGC-3′ and reverse 5′-TTTAATGTCACGCACGATTT-3′. The qPCR was performed on a 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) under the following thermal cycling conditions: Initial heating at 95°C for 5 min, followed by 35 cycles of 94°C for 30 sec, 60°C for 30 sec and 72°C for 30 sec, with a final dissociation stage at 95°C for 15 sec, 60°C for 30 sec and 95°C for 15 sec. The threshold cycle (Ct) value was defined as the number of PCR cycles in which the fluorescence signal exceeded the detection threshold value. The relative gene expression was analyzed using the 2^−ΔΔCt method.

At 72 h post-transfection, the THP-1 cells were lysed in cold lysis buffer (Beyotime Institute of Biotechnology, Beijing, China), centrifuged at 13,400 × g for 5 min at 4°C and the supernatant was collected. The protein concentration in this supernatant was determined using the Bradford method [bovine serum albumin (BSA) was used at 9 different concentrations (0.25, 0.5, 1, 1.5, 2, 3, 4, 5 and 6 mg/ml) to prepare a protein standard. After diluting the protein standards, the stock dye reagent was prepared (500 mg Coomassie Blue was dissolved in 500 ml methanol and was added to 100 ml phosphoric acid and 50 ml ddH₂O) that was diluted in 8 ml ddH₂O. Two milliliters of dye reagent were added to each tube of protein standard and incubated at room temperature for at least 5 min. The absorbance of the protein standards and experimental samples was carried out by spectrophotometry (Bausch and Lomb, Berlin, Germany) at 595 nm and, finally, a standard curve was plotted]; and the protein was separated using 10% SDS-PAGE (ST628; Beyotime Institute of Biotechnology), and then transferred onto a polyvinylidene difluoride membrane (Beyotime Institute of Biotechnology) using semi-dry transfer (POWER/RAC200; Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membranes were blocked at 4°C overnight in 5% skimmed milk in Tris-buffered saline containing 0.05% Tween-20 (TBST; ST825; Beyotime Institute of Biotechnology), and then incubated with rabbit anti-human AMFR polyclonal antibody (sc-33541) to identify the AMFR and AMFR-C proteins, rabbit anti-human ROCK2 polyclonal antibody (sc-5561), mouse anti-human cyclin D1 monoclonal antibody (sc-70899), mouse anti-human Bcl-2 monoclonal antibody (sc-7382) or goat anti-human β-actin polyclonal antibody (sc-1616) (all Santa Cruz Biotechnology, Inc.) at room temperature for 2 h. The membranes were then washed three times with TBST, and incubated with the appropriate horseradish-peroxidase-conjugated secondary antibody (Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China). The primary antibodies were diluted at 1:200. The secondary antibodies were diluted at 1:1,000. The membranes were developed using Western Blotting Luminol Reagent (sc-2048; Santa Cruz Biotechnology, Inc.), according to the manufacturer's instructions. Images were captured using Image Scanner III (Amersham Biosciences, Uppsala, Sweden).

The THP-1 cells were harvested 72 h after transfection and washed twice with PBS. The supernatant was discarded and replaced with 1 ml 70% cold methanol. Following incubation at 4°C for at least 12 h, the cells were washed with PBS and stained with 5% propidium iodide (PI; Beyotime Institute of Biotechnology), supplemented with RNase, and incubated in the dark at room temperature for 30 min. The DNA content was analyzed using flow cytometry (FACSCalibur; BD Biosciences, San Jose, CA, USA), with 10,000 events recorded for each sample. The percentage of cells in the G0/G1, S and G2/M phases were determined using CellQuest software (version 3.3; BD Biosciences).

At 72 h post-transfection, apoptosis was detected using an Annexin V-FITC Apoptosis Detection kit (Beyotime Institute of Biotechnology), according to the manufacturer's instructions. Briefly, the THP-1 cells were harvested, washed twice with cold PBS and resuspended in binding buffer (included in the Annexin V-FITC Apoptosis Detection kit) at 5×10⁵ cells/ml. Annexin V-FITC (5 µl) was added to 195 µl of the cell suspension, which was mixed and incubated for 10 min in the dark at room temperature. The cells were washed with binding buffer and centrifuged at 250 × g at room temperature. The supernatant was discarded and the cell pellet was resuspended in 190 µl binding buffer. PI (10 µl) was added, and the cells were vortexed and analyzed using flow cytometry (FACSCalibur; BD Biosciences), with 10,000 events recorded for each sample. Early apoptotic events were identified as annexin V-positive, PI-negative staining, determined using CellQuest software (BD Biosciences).

Data were analyzed using the SPSS 18.0 software package (SPSS, Inc., Chicago, IL, USA). Data are presented as the mean ± standard deviation from three independent experiments. Differences between groups were analyzed using an independent samples t-test or one-way analysis of variance, with a least significant difference test for post-hoc analysis. P<0.05 was considered to indicate a statistically significant difference.

The expression of AMFR and ROCK2 were detected in the THP-1 cells (Fig. 1A). At 72 h post-transfection with the pcDNA3.1-AMFR-C plasmid, AMFR expression was detected in the THP-1 cells transfected with pcDNA3.1-AMFR-C, but not in those transfected with the pcDNA3.1 control plasmid (Fig. 1B).

The mRNA expression of AMFR was significantly decreased in the THP-1 cells transfected with AMFR siRNA for 24, 48, 72 and 96 h, compared with those transfected with scrambled siRNA (P<0.05), and the lowest expression level of mRNA was measured at 72 h post-transfection with AMFR siRNA (Fig. 2A). The protein level of AMFR was also significantly decreased 72 h after AMFR siRNA transfection (P<0.05; Fig. 2B). No significant difference in the protein levels of AMFR were observed between the cells in the blank control and scrambled siRNA transfection groups (Fig. 2B).

The cell cycle was examined in the THP-1 cells transfected with AMFR-targeting siRNA using flow cytometry with PI staining. AMFR knockdown resulted in disruption of the cell transition between the G0/G1 phase and the S phase and, compared with the untransfected and scrambled siRNA-transfected cells, the percentage of cells in the G0/G1 phase was significantly increased and the percentage of cells in the S phase was significantly decreased in the AMFR siRNA transfected cells (P<0.05). However, there was no significant difference the fraction of cells in the G2/M phase (Fig. 3).

Apoptosis of the THP-1 cells was analyzed using annexin V/PI staining. The results revealed that the percentage of early apoptotic cells increased significantly from 3.88±1.43% of the untransfected THP-1 cells, and 4.01±1.52% of the scrambled siRNA-transfected cells to 19.58±4.29% of the AMFR siRNA-transfected cells (P<0.05; Fig. 4).

AMFR knockdown resulted in significantly decreased protein levels of detectable ROCK2, Bcl-2 and cyclin D1, compared with the untransfected and scrambled siRNA-transfected cells (P<0.05; Fig. 5A), whereas transfection with AMFR-C resulted in significantly increased protein levels of ROCK2, Bcl-2 and cyclin D1, compared with the cells transfected with the pcDNA3.1 plasmid (P<0.05; Fig. 5B).