Il 17 fitc

Mononuclear cells were immunostained with various combinations of the following fluorescence-conjugated antibodies: CD25-APC (eBioscience, San Diego, CA, USA), CD4-Paciffic blue (eBioscience), Foxp3-PE (eBioscience), H-2^d-percp-cy5.5 (eBioscience), IL-17-PE (eBioscience), Foxp3-APC (eBioscience), IFN-γ-APC (eBioscience), IL-4-PE (BD PharMingen), IL-17-FITC (eBioscience), H-2^b-FITC (eBioscience), IL-6-PE (Biolegend), and TNF-α-APC (BD). Before intracellular cytokine staining, cells were stimulated in culture medium containing phorbol myristate acetate (25 ng/mL; Sigma-Aldrich), ionomycin (250 ng/mL; Sigma-Aldrich), or monensin (GolgiStop, 1 μL/mL; BD PharMingen) in a 5% CO₂, 37°C incubator for 4 h. Intracellular staining was performed using an intracellular staining kit (eBioscience) according to the manufacturer’s protocol. Flow cytometry was performed on a FACSCalibur flow cytometer (BD PharMingen) with the FlowJo software (TreeStar, Ashland, OR, USA).

Cell pellets were prepared from the spleen tissues isolated from PIAS3- and Mock-treated mice. Populations of Th cells were examined by staining the tissues with a monoclonal Ab against CD4–peridin chlorophyll protein (perCP) and CD25-Allophycocyanin (APC) (eBioscience). Cells were permeabilized and fixed with CytoFix (BD Biosciences, San Jose, CA, USA) as instructed by the manufacturer, and stained with Abs against IL-17–FITC, IFNγ-PE, Foxp3-FITC (eBioscience), and PIAS3 APC (abcam, MA, USA). Flow cytometric analysis was primarily gated of CD4, then percentage of IL-17 positive, interferon-γ positive, Foxp3/CD25, and PIAS3 positive cells were calculated.

Chromatographically pure acetonitrile and methanol, ultrapure water, and other analytically pure reagents. Chlorogenic acid (Lot 171110; purity 99.37%); Forsythin (Lot 171103; purity 99.01%); Thymol (Lot 171210; purity 98.59%); Magnolol (Lot 171126; purity 99.29%), which were purchased from Shengshi Kangpu Chemical Technology Research Institute (Beijing, China). PrimeScript RT reagent kits (Lot: RR047A, TaKaRa, Japan), SYBR Premix EX Taq II (Lot: RR820A, TaKaRa, Japan). DEPC Water, (Sigma, USA); TLR7 Rabbit mAb (Lot: 2633S), MyD88 Rabbit mAb (Lot:4283S), NF-kappa B p65 Rabbit mAb (Lot: 8242S), GAPDH Rabbit mAb (Lot: 2118S) were purchased from CST, USA; The mouse lymphocyte separation solution, Goat anti-rabbit secondary antibody, BCA Protein Quantitative Kit, and potent ECL kits were all products of Multi Sciences Co., Ltd. in Hangzhou, China. CD4-PE (Phycoerythrin) (Lot: 4343463); CD25-APC (Allophycocyanin) (Lot: E07106-1634); IL-17-FITC (Lot: 4291420); Foxp3-PE-cyanine 5.5 (Lot: E16149-104), eBioscience™ Foxp3/Transcription Factor Staining Buffer Set Kit (Lot: 4343791) were purchased from eBioscience Inc., USA.