5 aza 2 deoxycytidine

KYSE-150 cells were incubated with different dosages of DNA methyltransferase inhibitor for 48 h before flow cytometry analysis. DNA methyltransferase inhibitor 5-Aza-2′-deoxycytidine was acquired from MedChemExpress (Monmouth Junction, NJ, USA) and fully dissolved in dimethylsufoxide (DMSO) at a concentration of 10 mM. Apoptosis was assessed using the Annexin V, 633 Apoptosis Detection Kit (Dojindo, Kumamoto, Japan). Cell cycle analysis was performed using the Cell Cycle and Apoptosis Analysis Kit (Beyotime; Jiangsu, China). Both assays were carried out and analyzed on a flow cytometer (Beckman Coulter) according to the manufacturer’s instructions. Data from the apoptosis assay were analyzed using FlowJo v10 (FlowJo, LLC). Cell cycle distributions were statistically determined by Modfit LT 3.2 software (Verity Software House; www.vsh.com; Topsham, ME).

T-cells were obtained from lung, spleen, thymus, or tracheobronchial lymph nodes (tLN). Mouse naïve CD4⁺ T-cells (CD4⁺CD62L^hiCD44⁻) were sorted by Naïve CD4⁺ T-cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Human CD4⁺ T-cells were sorted by CD4⁺ T-cell Isolation Kit (Miltenyi Biotec). The purity levels were above 95% detected by BD FACSCelestaTM. For in vitro experiments, mouse CD4⁺ T-cells were cultured in RPMI 1640 medium (Corning, Manassas, VA, USA) supplemented with 10% FBS, and 1% penicillin-streptomycin, and then stimulated by anti-CD3 (precoated, 5 μg/mL; BD Biosciences) and anti-CD28 (1 μg/mL; BD Biosciences) for 3 h. For T_H2 cell differentiation, naïve CD4⁺ T-cells were cultured under the following conditions: mIL2 (10 ng/mL; BD Bioscience), mIL4 (50 ng/mL; BD Bioscience), and anti-IFN-γ (10 μg/mL; BD Bioscience) with anti-CD3 (precoated, 5 mg/mL) plus anti-CD28 (1 mg/mL; BD Bioscience) for 5–7 days. Human CD4⁺ T-cells were stimulated by PMA (50 ng/mL; Sigma) and ION (500 ng/mL; Sigma) for 3 h. In pharmacologic inhibition experiments, T cells were pretreated with 5-aza-2′-deoxycytidine (1 μg/mL; MedChemExpress, Monmouth Junction, NJ, USA) and then stimulated with anti-CD3 (precoated, 5 μg/mL; BD Biosciences) and anti-CD28 (1 μg/mL; BD Biosciences) for 3 h.