Cell Culture and Stimulation
HMC-1 cells were cultured in IMDM with 100 U/ml of penicillin and streptomycin, and 10% fetal bovine serum (FBS) at 37 ̊C in 5% CO2. The HMC1 cells were treated with recombinant humanIL-35 (1-1000ng/ml) for 6 h. The cells were then stimulated with 50 nM of PMA plus 1 μM of A23187 and incubated at 37 ̊C for the indicated time periods (15 min-8 h).
Assay for the receptor of IL-35 (IL-12R-β2 and gp130)
The DNA was isolated from HMC-1 cells using RNA Reagent Kit (Tiangen Biological Manufacture Co. Ltd., Beijing, China). The primers for IL-12R-β2 and gp130were designed by ourselves. The forward primer sequence for IL-12R-β2 was 5' –AGAGGCGATGTGACTGTGAA–3’. The reverse primer sequence for IL-12R-β2 was 5'–TCAGGGGTGAGGTTGATTCC–3'. The forward primer sequence for gp130 was 5'–CCAGTGGTCACCTCACACTC–3'. The reverse primer sequence for gp130 was 5'–GGGCAAAATACCATCACCGC–3'. The conditions were 94°C for 90 s, and 94°C for 30 s and 55°C for 30 s of 35 cycles with a extension at 72°C for 5 min.
Cell viability
Cell Counting Kit-8 (CCK8, WST, China) was used for cell viability assay. Firstly, HMC-1 cells were seeded in a 96-wells plate at the density of 103 cells/well in 100 μl medium. Cells were treated with recombinant humanIL-35 (1, 10, 100, 1000 ng/ml) for 6 h at the beginning of test and cell viabilities were determined. In addition, cells were treated with different concentrations IL-35(1, 10, 50, 100, 1000 ng/ml) for 6 h prior to stimulation with 50 nM of PMA plus 1 μM of A23187 and incubated at 37 ̊C for 8 h. And then 10 μl CCK8 was added to each well and incubated for 1 h, and the optical density (OD) value was determined by microplate reader at 450 nm.
Histamine assay
Histamine levels from HMC-1 cells were determined using Human Histamine Elisa Assay Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturer’s instruction. The optical density(OD) was determined at 450 nm in a microplate reader.
Real-time quantitative PCR
HMC-1 cells were seeded in a 6-wells plate and then treated with recombinant humanIL-35(50 and 100 ng/ml) for 6 h prior to stimulation with 50 nM of PMA plus 1 μM of A23187 and incubated at 37 ̊C for 8 h. The mRNA levels of IL-4, IL-6, IL-17, IFN-γ and TNF-α in HMC-1 cells from different groups were determined by real-time quantitative PCR. Total RNA was extracted by Trizol reagent (Invitrogen Corp, Carlsbad, CA, USA) according to the manufacturer's instructions. The cDNA was synthesized from the total RNA by using RT reagent Kit with gDNA Eraser (Takara, Dalian, China). cDNA samples were amplified in a 20 μl reaction volume containing 10 μl of 2x SYBR GreenMaster Mix (Takara, Dalian, China), 2 μl of cDNA and 0.25 μM qPCR primers. The following primers were used: IL-4 (P216616, Bioneer, Inc., Daejeon, Korea); IL-6 (P211161, Bioneer, Inc., Daejeon, Korea); IL-17 (P291322, Bioneer, Inc., Daejeon, Korea); INF-γ (Catalog:HQP009467, GeneCopoeia, USA); TNF-α (P237423, Bioneer, Inc., Daejeon, Korea); GAPDH (5’-CGGAGTCAACGGATTTGGTC-3’ and 5’-CGGTGCCATGGAATTTGCCA-3’). The conditions were 95°C for 5 min, and 95°C for 15 s and 60°C for 30 s of 40 cycles with a final extension at 72°C for 5 min. The mRNA levels of IL-17 and IL-6were expressed as relative mRNA levels compared with control and determined by the 2-ΔΔCt method.
Western Blot Analysis
The expression of mitogen-activated protein kinases (MAPKs) in HMC-1 cells was measured by western blot. HMC-1 cells were treated with recombinant human IL-35(50 and 100 ng/ml) for 6 h prior to stimulation with 50 nM of PMA plus 1 μM of A23187 and incubated at 37 ̊C for 15 min. After treatment, cells were collected and lysed in ice-cold RIPA buffer (EMD Millipore, Billerica, Massachusetts) containing 1% phenyl- methylsulfonyl fluoride. The samples were vortex mixed for lysis for a few seconds every 15 minutes at 4 ̊Cfor 1 hour and centrifuged at 13,000 rpm for 50 minutes at 4 ̊C. Then, the samples were heated at 98 ̊C for 10 minutes and briefly cooled on ice. Then, total protein was extracted. Protein samples of 40 mg were electrophoresed on 12% Tris-glycine gels, subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis, and transferred to polyvinylidene fluoride membranes. Subsequently, membranes were incubated with primary antibody at 4 ̊C overnight and with the appropriate horseradish peroxidase-conjugated secondary antibody for 1 hour. The expression of MAPKs was determined with enhanced chemiluminescence reagents and exposure to a Kodak x-ray film (Eastman Kodak, Rochester, New York). The results were normalized to the expression of β-actin.
Statistical analysis
All data were expressed as mean ± SD. One-way analysis of variance, Mann–Whitney U Test or Wilcoxon sign-rank test were used to compare statistical differences between groups. Each experiment was carried out at least 3 times. P < 0.05 was set as the statistically significant.