The human skin cancer cell lines (A431 and SCC13) were obtained from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). All the cells were cultured as monolayers to 80% confluence in Dulbecco's modified Eagle's medium (DMEM; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 100 μg/ml penicillin-streptomycin (GE Healthcare, Logan, UT, USA) in a humidified incubator with 5% CO₂ at 37°C.

A431 and SCC13 cell lines were transfected with the cDNA of the BTG2 gene. For stable transfection, pcDNA3.1 expression vector (Thermo Fisher Scientific, Inc.) was used for insertion of BTG2 cDNA between the KpnI and BamHI sites. The orientation of the insertion was confirmed via restriction digestion and DNA sequencing. Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) was used to transfect the pcDNA3.1-BTG2 expression vector or the empty pcDNA3.1 vector into A431 and SCC13 cells, in accordance with the manufacturer's protocol. The cells were then cultured in DMEM containing 800 μg/ml G418 for antibiotic selection. After 28 days, positive clones were collected from the A431 and SCC13 cell cultures in the pcDNA3.1-BTG2 vector transfection group (A431-BTG2 and SCC13-BTG2) and the empty pcDNA3.1 vector transfection group (A431-PC and SCC13-PC). Western blot analysis was performed in order to detect BTG2 protein expression in the A431-BTG2, A431-PC, SCC13-BTG2 and SCC13-PC cell groups.

Transfected and untreated cells were seeded into 96-well culture plates (Corning Life Sciences, MA, USA) at a density of 1×10⁴ cells/well and cultured for 24 h with saturated humidity and 5% CO₂ at 37°C to form a cell monolayer. MTT solution (100 μl; Nalge Nunc International, Roskilde, Denmark) was aliquoted per well and incubated with 5% CO₂ at 37°C for 4 h. Next, the DMEM was discarded and 150 μl dimethyl sulfoxide was added to each well. The absorbance was measured at 570 nm with a microplate reader (BD Biosciences, San Jose, CA, USA).

When the cells reached 70–90% confluence, a serum-free DMEM was applied for synchronization. The cells were cultured for 24 h, then trypsinized (Sigma-Aldrich, St. Louis, MO, USA) and fixed with 100% ethanol overnight, followed by propidium iodide staining. Cell cycle analysis was performed after 30 min using a flow cytometer (BioVision, Inc., CA, USA). Each experiment was replicated three times.

Twenty-four well Transwell chambers (8 μm; Corning Life Sciences) were used to detect the invasive and migratory ability of the skin cancer cell lines. For the invasion assay, the upper chamber was washed with serum-free DMEM three times, filled with 20 μl diluted Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) and incubated for 30 min at 37°C to form the artificial basement membrane. Subsequently, all the cells were suspended in the serum-free DMEM and added to the upper chamber. The lower chamber was filled with DMEM containing 10% FBS. The cells were incubated for 24 h and those remaining in the upper chamber surface of the basement membrane were removed with cotton swabs, cells invading the lower chamber surface were stained with crystal violet. The number of cells crossing the polycarbonate membrane was counted under a microscope (CX22; Olympus Corporation, Tokyo, Japan), at a magnification of ×400. The migration assay was performed according to the above-mentioned procedure, except that Matrigel was not used, instead the Transwell chamber was used alone.

The cells were lysed using a lysis buffer (Sigma-Aldrich). The total protein (30 μg) was separated from each group by 10% SDS-PAGE (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and transferred onto polyvinylidene fluoride membranes (Thermo Fisher Scientific, Inc.). The membrane was blocked at room temperature for 1 h with Tris-buffered saline (TBS) containing 5% non-fat milk and then incubated overnight at 4°C with the mouse primary antibodies (all from Santa Cruz Biotechnology, Dallas, TX, USA) against BTG2 (1:2,500; cat. no. sc-517187), β-catenin (1:2,000; cat. no. sc-53484), cyclin D1 (1:3,000; cat. no. sc-20044), v-myc avian myelocytomatosis viral oncogene homolog (c-Myc; 1:2,500; cat. no. sc-47694) and β-actin (1:1,500; cat. no. sc-130300). After washing with TBS Tween-20 (TBST), the membrane was incubated for 1 h at 37°C with horseradish peroxidase-conjugated goat anti-mouse polyclonal antibody (1:3,000; Santa Cruz Biotechnology, Inc.; cat. no. 395760). Subsequent to another wash with TBST, protein expression was detected using an enhanced chemiluminescence kit (Bio-Rad Laboratories, Inc.).

Statistical analysis of experimental data was performed with SPSS 17.0 statistical analysis software (SPSS, Inc., Chicago, IL, USA). The Student's t-test was conducted for the comparison of two groups and one way analysis of variance was performed for multiple comparisons. Data are presented as the mean ± standard deviation and P<0.05 was considered to indicate a statistically significant difference.

BTG2 protein expression in A431 and SCC13 cells and their transfectants was detected by western blot analysis. BTG2 protein expression was observed in A431-BTG2 and SCC13-BTG2 cells; however, it was not detected in A431, A431-PC, SCC13 and SCC13-PC cells (Fig. 1).

The proliferation of A431-BTG2 cells was significantly reduced in comparison with the A431-PC and untreated A431 cells (P<0.05). No significant difference was identified between the control groups (A431 and A431-PC; Fig. 2A). Cell proliferation in the SCC13-BTG2 group was significantly reduced when compared with the control groups (P<0.05; Fig. 2B).

Cell cycle analysis of A431 cells in each group was performed using flow cytometry. BTG2 overexpression significantly increased the number of A431 and SCC13 cells in the G₀/G₁ phase; however, it decreased the number of A431 and SCC13 cells in the G₂/M phase (P<0.05; Fig. 3). These results suggested that cell cycle distribution in A431 and SCC13 cells was affected by increased BTG2 expression levels and resulted in cell cycle arrest in the G₀/G₁ phase in A431 and SCC13 cells.

The effects of BTG2 overexpression on invasion and migration of A431 and SCC13 cells were investigated. As shown in Fig. 4A and B, the invasive and migratory abilities of A431-BTG2 cells were significantly reduced when compared with A431-PC and untreated A431 cells (P<0.05). The effects of BTG2 overexpression on the invasion and migration of SCC13 cells were also determined. As shown in Fig. 4C and D, invasive and migratory abilities of SCC13-BTG2 cells were significantly reduced when compared with SCC13-PC and untreated SCC13 cells (P<0.05).

Western blot analysis was performed to detect the effects of BTG2 overexpression on β-catenin, cyclin D1 and c-Myc. A significant decrease in the expression levels of β-catenin, cyclin D1 and c-Myc in A431-BTG2 cells when compared with A431-PC and untreated A431 cells (P<0.05; Fig. 5).