Rabbit polyclonal antibodies against CLDN2 (ab107059) and anti-human β-actin (ab8226) were purchased from Abcam (Massachusetts, US). Rabbit anti-human phospho-ERK1/2 (#9251), rabbit anti-human ERK1/2 (#4695), rabbit anti-human ras (#14429), rabbit anti-human raf-1 (#2330), rabbit anti-human phospho-MEK1/2 (#2338), rabbit anti-human MEK1/2(#8727) and rabbit anti-human afadin (#13531) were purchased from Cell Signaling Technology (Boston, USA) and a streptavidin-peroxidase immunohistochemistry reagent kit was purchased from Maixin Biology (Fujian, China).
Western blotting
The protein concentration of cell lysates was determined using a bicinchoninic acid (BCA) Protein Assay Kit (Pierce Chemical Co., Rockford, Illinois, USA). Then, 30 µg of total protein was separated via 10% SDS-PAGE and transferred onto nitrocellulose membranes (Millipore, Temecula, California, USA). Next, the membrane was blocked and investigated with rabbit anti-human phospho-ERK1/2 antibody, rabbit anti-human ERK1/2 antibody, rabbit anti-human p-MEK1/2 antibody, rabbit anti-human MEK1/2 antibody, rabbit anti-human CLDN2 antibody, mouse anti-human β-actin antibody, and rabbit anti-human afadin antibody at a 1:1000 dilution at 4 °C for 12 h. After three washes with phosphate-buffered saline (PBS), the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnologies, California, USA) at a 1:1000 dilution at room temperature (RT) for 30 min. Immunoreactive bands were detected using ECL western blotting reagents (GE, Fairfield, Connecticut, USA) and analyzed with Image Lab 6.0.1 Software from Bio-Rad Laboratories.
Immunofluorescence method
Cells were washed thrice with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 10 min at RT, permeabilized with 0.1% Triton X-100 (Sigma-Aldrich; #9002-93-1) for 10 min at RT and blocked in 2% bovine serum albumin (Bote Biotechnological Corporation, Shandong, China) in PBS for 1 h at RT. Staining with primary antibodies was performed with rabbit anti-human CLDN2 and rabbit anti-human afadin antibodies, which was diluted in blocking solution (1:1000 dilution) for 30 min at RT. The cells were incubated with Alexa Fluor®647-conjugated anti-rabbit IgG antibody (ab150093, Santa Cruz Biotechnologies, California, USA) at a 1:1000 dilution. Images were taken using an Olympus IX81 microscope with an MT20/20 illumination system.
Immunoprecipitation
Cells in the logarithmic growth phase were harvested and lysed in 600 μl of pre-chilled protein lysis buffer (4 °C) for 30 min. The lysate was centrifuged at 14,000 rpm at 4 °C for 20 min, after which the protein concentration was determined with a Beyotime protein assay kit. The protein supernatant was divided into two volumes. One 50-μl volume was mixed with 5× protein loading buffer, boiled in a water bath for 10 min, and then was stored at − 20 °C as the input control. The remaining volume of lysate was mixed with 30 µl of protein A/G agarose beads and 2 µg of a normal IgG antibody of the same species as the IgG used for immunoprecipitation. The mixture was rotated at 4 °C for 1 h and then centrifuged at 2500 rpm at 4 °C for 5 min, after which the supernatant was transferred to a new centrifuge tube. Next, 2 µg of the primary immunoprecipitation antibody (rabbit anti-human raf-1 antibody) was added to the total protein, and the mixture was adjusted to a final volume of 600 μl with protein lysis buffer. The mixture was slowly mixed overnight at 4 °C on a shaker. Forty microliters of adequately mixed protein A/G agarose beads was added to the mixture, which was slowly rotated at 4 °C for 90 min and then centrifuged at 3000 rpm at 4 °C for 10 min. Afterwards, the supernatant was removed, and the protein A/G agarose pellet was saved. Protein lysis buffer was used to wash the protein A/G agarose pellet five times, after which 25 μl of protein lysis buffer was added to resuspend the pellet followed by the addition of 5× loading buffer. The mixture was boiled for 5–10 min and used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Cell counting kit-8 assay
A cell proliferation curve was determined by the colorimetric water-soluble tetrazolium salt assay (Cell counting kit-8; Dojindo, Kumamoto, Japan) according to the manufacturer’s protocol. The cells were seeded into 96-well plates in triplicate, and cell proliferation was recorded every 12 h for 4 days.
Colony formation assays
The experimental method was the same as describe previously [21].
Flow cytometry cell cycle analysis
The experimental method was the same as describe previously [21].
Wound-healing assay
Cells were cultured in a monolayer at 70% confluence on gridded plastic dishes. Monolayers were wounded by scratching with a 100-μl pipette tip and subsequently washed 3 times with phosphate buffer saline. Images of the wound site were taken using a light microscope (E100, Nikon Instruments Inc, Japan) (magnification 200×) at the same location at 0, 12 h and 24 h, respectively.
Transwell chamber method
The cells were grown in a monolayer at 90% convergence and were maintained in FBS-free medium for 12 h. Matrigel (BD Biosciences, cat. no. 356234) was added to the upper Boyden chamber (Millipore, Bedford, MA) in 24-well plates and the plates were maintained in a cell incubator at 37 °C for 15 min. Then, medium containing chemotactic factors, which had been collected from the cell culture, was added to the 24-well plate. The cells were supplemented with Matrigel and cultured in a cell incubator at 37 °C for 6 h.
Short hairpin RNA (shRNA) method
Frozen glycerol bacterial stocks containing pGCSIL-scramble, pGCSIL-CLDN2-RNAi and pGCSIL-afadin-RNAi frozen glycerol bacterial stocks were purchased from Nanjing KeyGen Biotech Co, Ltd. HEK 293T cells (0.2 × 107) were seeded and maintained for 24 h to achieve 70–80% confluence in 6-well dishes (Costar, Cam- bridge, MA). The plasmids, including 10 μg pGCSIL-afadin-RNAi, pGCSIL-CLDN2-RNAi or pGCSIL-scramble, 5 μg packaging vector pHelper 1.0 and 5 μg vesicular stomatitis virus glycoprotein (VSVG) expression plasmid vector, were supplemented with Opti-MEM and 1.0 ml. A total of 50 μl of Lipofectamine was added to 950 μl FBS-free medium. These two mixtures were mixed and added to the cells. Lentiviral particles were harvested 48 h after transfection, and the viral titer was determined by counting green fluorescent protein (GFP)-expressing cells under fluorescence microscopy (Nikon Diaphot 300®) with filters 96 h after transfection.
Patients and tissue samples
Biopsies were gathered from 27 patients with a pathologically confirmed diagnosis of OS who received treatment at the Qilu Hospital of Shandong University between June 2007 and May 2012. The patients were chosen based on the following criteria: no history of radiotherapy, no history of chemotherapy, and no prior malignant disease. The grade and classification of OS patients were referred to the American Joint Committee on Cancer (AJCC) tumor node metastasis (TNM) staging system. 3 of the biopsies were taken from the tibia, 3 from the humerus, 16 from the femur, 2 from the fibula, and 1 each from the forearm, the hand, and the pelvis. 21 of the cases also exhibited pulmonary metastasis. Bone tissue that was identified as histologically non-neoplastic was also obtained from 10 patients with osteoarthritis who were treated at the Qilu Hospital of Shandong University between October 2006 and September 2011. There were 6 men and 4 women with an average age of 47 years.
Immunohistochemistry
Immunohistochemistry was used to detect the expression levels of CLDN2 and afadin in paraffin-embedded biopsy specimens from 27 patients who had a diagnosed primary OS and had not undergone chemotherapy or radiotherapy before biopsy. The experimental method was the same as described previously [21], and the antibodies used were rabbit anti-human CLDN2 and afadin antibody. Negative control slides were incubated with isotype antibodies at same dilution with primary anti-human CLDN2 and afadin antibody. The expression levels of CLDN2 and afadin located at the cell membrane and cytoplasm were taken as positive. The staining and scoring of the CLDN2 and afadin protein expression levels were classified semi-quantitatively based on the total combined scores of the percentage of positively stained tumor cells together with the staining intensity as previously described [22]. The final score of the protein expression was defined as ‘low’ if < 30% of tumor cells stained positive, and ‘high’ if > 30% of tumor cells stained positive. At least five different areas of the tumor were examined, and the mean of the results was used as the final expression score in each case.
Statistical methods
All of the experiments were repeated three times, and all of the data are based on the mean ± SD of at least three experimental results. The Chi-square test/Chi-square goodness of fit test was used to analyze the correlations between CLDN2 and afadin expression and clinical pathological indicators. The results were analyzed by Student’s t-test, and P < 0.05 was considered to be statistically significant.