37uC for 30 min in 7-amino-actinomycin D (7-AAD) staining solution (BD Biosciences Pharmingen). Cell counts in each phase of the cell cycle were estimated using a FACSCalibur (Beckton Dickinson) and Cellquest 3.0 software. Western blotting analysis of IL-22R on the surface of cells A498 cells (16106) were seeded into 6-well plates and cultured for 6 hours in serum-free medium. Total cell lysates were prepared on ice in a buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholic acid, 0.1% SDS and 1 mM EDTA. Proteins were isolated by 10% SDS-PAGE and the concentration of protein was assessed using the Bradford dye- binding protein assay (Bio-Rad, Richmond, CA, USA), and SDS- polyacrylamide gel electrophoresis was performed. Mouse IL-22 Ra1 antibody was purchased from R&D system. Anti- b-actin monoclonal antibody (Abcam, Cambridge, UK) was used as an internal loading control. The immune complexes were detected using the ECL plus Western Blotting Detection System (Amer- sham, Aylesbury, UK). Hepatoma cell line HepG2, purchased from Cell Bank of China Union Medical University (Beijing, China) and cultured as A498 cells, was served as positive control of IL-22R. As for negative control, we selected human B cells purified from human spleen. The splenic tissue was gathered from a patient with informed consent who was performed splenectomy due to splenic rupture. The procedure of B cells purification was followed the statement of Le Aˆcart [43]. The purified B cells populations were confirmed CD19+ .98% by flow cytometry assay. The detection procedure of IL-22R on the surface of these cells was similar as showed above. Western blotting analysis of p-STAT1 and p-ERK1/2 after IL-22 exposure A498 cells (16106) were seeded onto 6-well plates and cultured for 6 hours in serum-free medium. Cells were then treated with 10 ng/ml, 20 ng/ml, 50 ng/ml and 100 ng/ml rhIL-22 for 5 min, 10 min, 15 min, 20 min, and 30 min respectively or with PBS as control. Total cell lysates were prepared in a buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholic acid, 0.1% SDS and 1 mM EDTA on ice. Samples were sonicated for 30 seconds and boiled at 95uC for 5 min. Proteins were isolated by 10% SDS-PAGE and the concentration of protein was assessed using the Bradford dye- binding protein assay (Bio-Rad, Richmond, CA, USA), and SDS- polyacrylamide gel electrophoresis was performed. phospho- STAT1 (Tyr701)/STAT1 (9H2) antibody and phspho-ERK1/2 (Thr202/Tyr204)/MAPK (Erk1/2) antibody were purchased from Cell Signaling. Anti- b-actin monoclonal antibody (Abcam, Cambridge, UK) was used as an internal loading control. The immune complexes were detected using the ECL plus Western Blotting Detection System (Amersham, Aylesbury, UK). STAT1 small interfering RNA STAT1 small interfering RNA (siRNA) sequences were used to removal the contribution of STAT1 on cells growth. STAT1 siRNA sense sequence r(CACGAGACCAAUGGUGUGG)d(TT) or STAT1 target siRNA anti-sense sequence r(CCACACCAUUG- GUCUCGUG) d(TT) ( all from Qiagen) was used to knock down STAT1 expression (DNA target sequence: AAC ACG AGA CCA ATG GTG TGG; GenBank accession no. NM-139266, nucleo- tides 893–913). A scrambled sequence was used as a negative control sequence. The STAT1 siRNA transfection was per- formed following the Qiagen TransMessenger transfection protocol Interleukin22 Suppress Renal Cell Carcinoma Growth (Qiagen Inc, Valencia, CA). Cells were transfected with siRNA and incubated for 48 hrs. The same dose of IL-22 treated siRNA transfected A-498 cells as above shows and proteins expression such as STAT1, p-STAT1 were assessed by Western blot assay. Growth inhibition activity of IL-22 on A498 cells transfected with STAT1 siRNA. A498 cells transfected with STAT1 siRNA was used to confirm the role of STAT1 in cell proliferation after treated with IL-22. The 1,000 cells/well of STAT1 siRNA A498 cells were exposure to IL-22 with the same concentration gradient. The growth inhibition assay was performed as A498 cells’. Flow cytometric analysis of apoptosis A498 cells (16106 /well) were seeded onto 6-well plates. After allowing 6 hours for the cells to adhere, the cells were treated with rhIL-22 (with the dose of 25, 50, 100, 200 and 400 ng/ml respectively) for 24 or 72 hrs respectively. The cells were then trypsinized, washed with binding buffer, and incubated with FITC- labeled annexin V antibody for about 20 min at 37uC in the dark. The cells were resuspended and 1% FCS and 10 ml of propidium iodide (PI)3 solution (1 mg/ml) were added. Flow cytometry was performed in a FACScan with a single cell gate (BD Biosciences). The dot plots in quadrants quantified the percentage of cells. Western blotting analysis of IFN-a and TNF-a in A498 xenografts. The tumor tissues were harvested after the mice were sacrificed and the samples were immediately frozen and crushed into powder in liquid nitrogen. Ice-cold lysis buffer (T- PER, Pierce) with protease inhibitor (Complete Mini, Roche) was added and then kept on ice for 30 min. Proteins were isolated by 10% SDS-PAGE and the protein concentration was estimated by Bradford dye-binding protein assay (Bio-Rad, Richmond, CA, USA) in accordance with the manufacturer’s protocol. The protein (30 mg) were dissolved in 10–12% polyacrylamide- SDS gels and transferred onto a nitrocellulose membrane. The membrane was immersed in blocking buffer (5% non-fat dry milk/ 1% Tween 20 in 20 mM TBS, pH 7.5) for 1.5 h at room temperature and incubated with the appropriate primary antibod- ies, mouse monoclonal anti-TNF-a (1: 50) and IFN-a (1:100) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in blocking buffer overnight at 4uC, followed by incubation with complementary secondary antibody. Proteins were detected by the ECL plus Western Blotting Detection System (Amersham, Aylesbury, UK). Statistical analysis. All experiments were carried out in triplicate, and continuous variables are expressed as mean 6 SD. For statistical analysis, continuous variables that met the criteria for a normal distribution were determined using a two-tailed Student’s t-test. Statistical analysis of the results of the flow cytometry experiments was performed using the Fisher’s exact test and two-sided tests. The x2 test was used for numeration data. Significance for all tests was established at P,0.05. Supporting Information Figure S1 Schematic diagram for the mechanism of IL- 22 effect on A498 cells. After IL-22 bind with IL-22R and IL- 10R2, the IL-22 receipt compound is formed and the activation of STAT1 and deactivation of ERK1/2 pathway were followed, which results the growth inhibition of A498 cells. (TIF) Author Contributions Conceived and designed the experiments: FZ DS. Performed the experiments: DS. Analyzed the data: DS. Contributed reagents/materi- als/analysis tools: YZ. Wrote the paper: FZ YT. PLoS ONE | www.plosone.org 7 May 2011 | Volume 6 | Issue 5 | e20382