Real-time RT-PCR and western blotting were used to evaluate target molecule expression. both and < 0.01 compared with the saline group. SqC immunotherapy The SqC-bearing mice were treated (intraperitoneal (ip) injection) with SEB (2 g/mouse/day) and/or SqCAg (SqC antigen (the protein extracts of SCC VII cells; 50 g/mouse/day)). A group of SqC-bearing mice were treated with SEB/SqCAg and the anti-IL-9 neutralizing antibody (100 g/mouse; ip on days 8, 12, and 16). Another group of SqC-bearing mice were treated with SEB/SqCAg and a control antibody (IgG isotype, 100 g/mouse; ip on days 8, 12, and 16). Enzyme-linked immunosorbent assay (ELISA) The cytokine levels of IL-4, IFN-, IL-9, IL-10, and Foxp3 were determined by ELISA with commercial reagent kits, following the manufacturer's instructions. Cell isolation from SqC tissue The SqC tissue was excised from SqC-bearing mice and cut into small pieces (approximately 2 2 2 mm) and incubated with collagenase IV (0.5 mg mL?1) for 2 h at 37?C with moderate agitation. The cells were exceeded through a cell strainer (40 m) and collected by centrifugation at 1500 rpm for 5 min. A portion of the single cell suspension was analyzed by flow cytometry. For SqC cell isolation, T cells, B cells, DCs, mast cells, eosinophils, fibrocytes, and NK cells were selected by MACS; the remaining cells were used as SqC cells in other experiments. Flow cytometry CD4+ T cells(106 cells/sample) were blocked with 1% bovine serum albumin (BSA) for 30 min and incubated with Colchicine fluorochrome-labeled antibodies of interest or the IgG isotype (used as a negative staining control). For intracellular staining, the cells were fixed and permeabilized for 2 h and then stained with the fluorochrome-labeled antibodies of interest or the IgG isotype. After washing, the cells were analyzed by flow cytometry. At least 100 000 cells were analyzed for each sample. The data were analyzed with the Flowjo software,withthe IgG isotype-stained cells as the gating reference. T-cell proliferation assay CD4+CD25? T cells were isolated from the SqC tissue or the spleen by MACS and labeled with carboxyfluorescein succinimidyl amino ester (CFSE). The cells were cultured in the presence of phorbol-12-myristate-13-acetate (PMA; 40 ng mL?1), DCs (T cell: DC = 5:1), and specific antigen (Ag; the SqC cell extracts; 5 g mL?1). Three days later, the cells were analyzed by the CFSE-dilution assay in a flow cytometer (FACSCanto II, BD Biosciences, Shanghai, China). Western blotting The cells were lysed for western blotting in a protein lysis buffer. Nuclear extracts were obtained using a NE-PER cell fractionation kit (Thermo Scientific, Shanghai, China). The cell lysates or nuclear Colchicine proteins were fractionated on a 12.5% Colchicine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% skim milk for 30 min, incubated with the primaryantibodies (0.2 g mL?1) overnight at 4?C, and then incubated with the secondary antibodies (labeled with horseradish peroxidase) for 1 h. Washes with Tris-buffered saline with Tween 20 were performed after each incubation. The immunoblots around the membrane were developed with an enhanced chemiluminescence kit. The results were photographed with a KODAK Image Station 4000Pro (KODAK, Shanghai, China). Co-immunoprecipitation analysis (Co-IP) The cells were lysed in Co-IP buffer; then, the lysates were precleared with 50 L of pansorbin cells (Calbiochem, Shanghai, China) for 2 h, and this was followed by centrifugation. The samples were precleared by incubation with protein G agarose beads for 2 h at 4?C. Two micrograms of anti-PU.1, anti-STAT5, anti-HDAC1, or the IgG isotype (unfavorable control) was added to the precleared lysates in the presence of protein CLTA G agarose beads and incubated at 4?C overnight. The immune complexes around the beads were eluted with an elution buffer and separated by SDS-PAGE, transferred to a PVDF membrane, and immunoblotted with antibodies targeting PU.1, STAT5, or HDAC1. The subsequent analysis followed the western blotting procedures. Real-time quantitative PCR (RT-qPCR) Total RNA was extracted from CD4+ T cells using TRIzol reagent. The cDNA was synthesized with a Reverse Transcription Kit according to the manufacturer’s instructions. The PCR was performed with a Bio-Rad MiniOpticon Real-Time PCR System using the SYBR Green.