Cytosolic nuclear fractionation extraction kit

Human ESCC cell line KYSE150 was purchased from Shanghai Zhongqiaoxinzhou Biotechnology (Shanghai, China). Cell lines of KYSE510, TE-1, Eca-109, KYSE180, and Het-1A were maintained in the laboratory of thoracic surgery of West China Hospital, Sichuan University. All ESCC cells were cultured in RPMI-1640 (Gibco, USA), and Het-1A cells was cultured in DMEM (Gibco, USA), supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin, in an incubator with 5% CO₂ at 37 °C. The transfection was performed with jetPRIME^™ (Polyplus-transfection, France) according to the manufacturer’s instruction. For construction of PIWIL2 knockdown stable cells derived from KYSE150, transfected cells were selected at 72 h after transfection by treatment of 1 mg/ml G418 for 3–4 weeks. The stable cells lines were further identified by detecting PIWIL2 expression by Western blotting. To isolate the cytoplasmic component from the nuclear one, cells were treated with the cytosolic (C)/nuclear (N) fractionation extraction kit (Beyotime, China) according to the manufacturer’s instructions.

To isolate the cytoplasmic component from the nuclear one, cells were treated with the cytosolic (C)/nuclear (N) fractionation extraction kit (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instructions.