P237423

HMC-1 cells were seeded in a 6-wells plate and then treated with recombinant human IL-35 (50 and 100 ng/ml) for 6 h before 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 2× 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.

The mRNA levels of IL-6, TNF-α, IL-17, IL-4, IFN-γ, IL-10, and GAPDH in PBMCs after incubated with IL-29 or PBS and the expression of IL-6, IL-8 and GAPDH in HaCat cells after incubated with IL-29, TNF-α or PBS was determined by real-time quantitative PCR. 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 2 × SYBR Green Master 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-10 (P285360, Bioneer, Inc., Daejeon, Korea); IL-17 (P291322, Bioneer, Inc., Daejeon, Korea); IFN-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 the 30 s of 40 cycles with a final extension at 72 °C for 5 min. The mRNA levels of IL-6, TNF-α, IL-17, IL-4, IFN-γ, IL-10, and IL-8 were expressed as relative mRNA levels compared with control and determined by the 2-ΔΔCt method.