Ha suz12

HEK293T cells were cultured in DMEM (Gibco) supplemented with 10% fetal calf serum, 100 U/ml of penicillin, 100 U/ml of streptomycin. Flag‐Brd4, HA‐Ezh2, HA‐EED, and HA‐Suz12 plasmids were purchased from Addgene. The cDNA encoding Foxp3 was cloned by PCR and ligated to pWPXLd expression vector. Lentiviral vectors encoding murine Brd2, Brd4, Foxp3, Fbxw7, or negative control shRNA were ligated with BamHI and EcoRI into the pLVshRNA‐EGFP‐Puro. PEI were used for transfection in HEK293T cells. For lentivirus‐mediated gene transfer to naïve CD4⁺ T cells, HEK293T cells were transfected with lentiviral plasmids. After 6 h, culture supernatant containing lentivirus was collected 48 and 72 h after transfection. The supernatant was filtrated and concentrated, supplemented with 5 μg/ml polybrene and were used to spin‐infect naïve CD4⁺ T cells activated with anti‐CD3 and anti‐CD28. Spin infection was performed at 500 g for 90 min at room temperature. Cells were then cultured at 37°C for 4 h, and cytokines were added for polarization.