The RPMI 1640 medium, Dulbecco’s modified Eagle’s medium (DMEM), liposome Lipofectamin 2000, and Trizol reagent were obtained from Invitrogen (Carlsbad, CA, USA). Mouse monoclonal anti-HBx antibody, Mouse monoclonal anti-LASP-1 antibody, Immobilon Western Chemiluminescent HRP Substrate were from millipore (Temecula, CA, USA), rabbit polyclonal anti-phospho-Akt (Ser-473) antibody was from Bioword Technology (Atlanta, GA, USA), rabbit polyclonal anti-Akt antibody, goat anti-mouse IgG-HRP, goat anti-rabbit IgG-HRP were from Santa Cruz (CA, USA), Mouse monoclonal anti-GAPDH antibody, DAPI were from Beyotime Institute of Biotechnology (Jiangsu, China). FITC-conjugated secondary antibody was from Zhongshan Goldenbridge Biotechnology (Beijing, China). Cell counting kit-8 was from Dojindo Laboratories (Kumamoto, Japan). Transwell cluster plates (8.0 μm pore, 24-well) were from Corning Costar (Cambridge, Massachusetts, USA). Primers were synthesized by Shanghai Sangon Biological Engineering Technology and Services (Shanghai, China). TIANScript RT Kit was from TIANGEN Biotech (Beijing, China). G418 was purchased from Promega (Madison, WI, USA), A 50 mg/ml stock solution of G418 was prepared in 100 mM HEPES (pH 7.3) and stored at 4°C. LY294002 was obtained from Sigma (St. Louis, MO, USA). A 100 μM stock solution of LY294002 was prepared in dimethyl sulfoxide (DMSO) and stored at 4°C in the dark. Treatment concentration of LY294002 was prepared fresh for each experiment by serial dilution into 0.01% DMSO in RPMI 1640 medium or in DMEM. All other chemicals and reagents were of analytical grade.
Cell culture and stable transfection
Human hepatocarcinoma cell line, HepG2 cell, Huh-7 cell, obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), were cultured in RPMI 1640 medium or in DMEM supplemented with 100 mL/L fetal bovine serum at 37°C in 5% CO2. When the cell fusion rate reached 80%, in the presence of the liposome Lipofectamin2000 according to the manufacturer’s instructions, HepG2 cells and Huh-7 cells were transfected with plasmid pcDNA3.1-X, which contains the full length HBX sequence, was constructed in mammalian expression vector pcDNA3.1 (Invitrogen, Carlsbad, CA, USA) as described previously. Forty-eight hours post-transfection, the transfected cells were incubated in selection medium containing 800 mg/ml G418. Stable cell lines, named HepG2-HBX and Huh-7-HBX cells respectively, were selected after formation of resistant clones.
The total RNAs of HepG2-HBX, Huh-7-HBX and control cells were prepared with Trizol reagent by manufacturer’s instructions. The reverse transcription was performed with TIANScript RT Kit. Primer sequences used for HBX were TGTGAAGCTTATGGCTGCTAGGC and TGTGGAATTCTTAGGCAGAGGTG. Primers for LASP-1 were GTGTATCCCACGGAGAAGGT and TGCCA CTACGCTGAAACCT. Primers for β-actin were GGCATCGTGATGGACTCCG and GCTGGAAGGTGGACAGCGA. The amplification condition was 94°C for 45 s, 58°C (between 55°C to 60°C) for 35 s, 72°C for 1 min for the 35 cycles and a final extension at 72°C for 5 min each. The PCR products were subjected to electrophoresis in 1% agarose gel and visualized by ethidium bromide staining.
Western blot analysis
For protein extracts, cells were lysed in cell lysis buffer [20 mM Tris PH7.5, 150 mM NaCl, 1% TritonX-100, 2.5 mM sodium pyrophosphate, 1 mM EDTA, 1%Na3VO4, 0.5 μg/ml leupeptin and other phosphatase inhibitors, 1 mM phenylmethanesulfonyl fluoride (PMSF)]. The lysates were collected by scraping from the plates, and then centrifuged at 10,000 × g at 4°C for 5 min. The protein concentration was measured by BCA Protein Assay Kit and adjusted at equal pace. 60 μg total protein was subjected to SDS-PAGE and transferred onto PVDF membranes, the membranes were blocked with 3% BSA in TBS containing 0.01% Tween-20 for 3 h at room temperature, and then incubated with specific primary antibodies: mouse monoclonal anti-HBx antibody (1:500), mouse monoclonal anti-LASP-1 antibody (1:2000), rabbit polyclonal anti-phospho-Akt (1:500), goat polyclonal anti-Akt antibody (1:500) and mouse monoclonal anti-GAPDH antibody (1:3000), respectively, overnight at 4°C. Then, the membranes were incubated with goat anti-mouse IgG-HRP (1:2000), goat anti-rabbit IgG-HRP (1:2000), rabbit anti-goat IgG-HRP (1:2000), separately, for 3 h at room temperature. The protein bands were detected with Immobilon Western Chemiluminescent HRP Substrate.
The cells were seeded on coverslips at a density of 1 × 105 cells/coverslip and incubated for 24 h. Then the coverslips were washed with PBS, fixed with ice-cold acetone for 10 min. After blocked with 3% bovine serum albumin in PBS for 30 min. the coverslips were incubated with mouse anti-LASP-1 (1:200) at room temperature for 2 h and washed three times with PBS for 10 min/wash. Incubation followed for 30 min at room temperature with FITC-conjugated secondary antibody (1:50). Nuclei were stained using DAPI for 10 min. Then the coverslips were washed with PBS for 10 min/wash. Micrographs were acquired by using an Olympus BX51 fluorescence microscope at × 400 magnification.
siRNA preparation and transfection
Expression of human LASP-1 was knocked down with siRNA targeting the sequence CACGCCCGAGCTCCAGAGAAT. The negative control siRNA targeting an unknown mRNA sequence GTCTCCACGCGCAGTACATTT was used as a control. Both siRNAs were synthesized by Invitrogen using pcDNA™6.2-GW/EmGFP-miR vector. A blast search against the complete human genome verified that the selected sequences were specific for the target gene. Transient transfection of siRNA was also mediated by liposome Lipofectamine 2000 reagents and carried out as described in the manufacturer’s instructions.
Cell viability assay
The cells were prepared at a concentration of 3 × 104 cells/ml, and 100 μl of cell suspension were placed into 96-well plates (5 replica wells for each cell line). After incubation for 1, 2, 3 and 4 days, the number of viable cells was analyzed using the Cell Counting Kit-8 (CCK-8) following the manufacturer’s instructions. The optical absorbance at wavelength of 450 nm was measured in a plate reader (ClinBio-128, SLT, Austria).
Plate clone formation assay
About 200 cells were added to each well of a 6-well culture plate, and each group contained three wells. After incubation at 37°C for 14 days, the cells were washed twice with PBS and stained with Crystal Violet Staining Solution. The number of colonies containing 50 cells was counted under a microscope and the plate clone formation efficiency was calculated using the formula: plate clone formation efficiency = (number of colonies/ number of cells inoculated) × 100%.
Flow cytometry analysis
After transfection with LASP-1 siRNA for 48 h, 4 × 105 cells were collected, washed twice with PBS and incubated with 1 ml of 75% cold alcohol at 4°C overnight. After washed three times with PBS, the cells were stained with 0.5 ml propidium iodide (PI) solution (BectonDickinson, USA) for 30 min at 37°C in the darkroom. The cell cycle distributions were then measured flow cytometers (FACSCalibur, BectonDickinson, USA), and the results were analyzed by MODFIT 3.0 software (BectonDickinson, USA).
For transwell migration assay, 6 × 104 cells in serum-free medium were seeded in each cell culture insert, which contains a polyethylene terephthalate membrane (6.5 mm in diameter, 8 μm pore size). The bottom chamber was prepared with 10% FBS as a chemoattractant. Cells were incubated at 37°C for 24 h. Nonmigrated cells were scraped off the upper surface of the membrane with a cotton swab. Migrated cells were fixed by 4% paraformaldehyde and stained with crystal violet Staining Solution for photography. For quantification, the cells were counted under a microscope at × 400 magnification in five randomly selected fields.
Wound healing assay
For wound healing assay, the cells were seeded at 2.0 × 105 cells/well in 24-well plates and allowed to reach 100% confluence. A scratch wound was created on the cell surface using a micropipette tip. The wound area was photographed by bright-field microscopy at × 100 magnification at different time points after wounding. The width of the wound was measured and the migration distance was calculated as the formula: migration distance = (wound width at the beginning-wound width after treatment) / 2 (μm). Three separate visual fields were measured in each experiment.
All experiments were performed three times. Semiquantitative analysis of the bands was measured with the Image J analysis software (Version 1.30v, Wayne Rasband, NIH, USA). The data were presented in the mean ± SD format and analyzed by independent-Samples T Test or one-way ANOVA (SPSS version13.0), P < 0.05 was considered statistically significant.