The 293T cells were cultivated in the medium composed of Dulbecco’s modified Eagle medium (DMEM) (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 2% penicillin/streptomycin (Gibco), maintained at 37 °C in 5% CO2. The process for lentivirus generation of shRNA was performed as previously reported [22 (link)]. Briefly, when the confluence of cultured 293T cells reached 80%, pLKO.1-shSOCS1, psPAX2 and pMD2.G were co-transfected into 293T cells through TransIntro™ EL Transfection Reagent (TransGen Biotech, Beijing, China) for generating lentiviral particles (shSOCS1) in accordance with the manufacturer’s instructions. The 293T cells were co-transfected with pLKO.1-TRC, psPAX2 and pMD2G as the non-interfered control group (shNC). They were all measured by the serial dilution method for the infection titer of concentrated lentivirus particles.
Buffalo mammary epithelial cells (BuMECs) were isolated from the mammary gland tissue of lactating Binglangjiang buffalo (60 d postpartum) and purified based on the differential sensitivity of the cells to trypsin digestion as previously described by our group [22 (link),23 (link)]. The BuMECs purified and identified by cytokeratin 18 (Sigma, Louis, MO, USA) were cultivated and expanded to passage five in a basal medium composed of DMEM (Gibco), 10% fetal bovine serum (Gibco), 100 μg/mL penicillin/streptomycin (Gibco) and various cytokines, including 5 μg/mL insulin (Sigma), 2 μg/mL hydrocortisone (Sigma) and 100 ng/mL epidermal growth factor (Sigma), and maintained at 37 °C under 5% CO2. When the cell confluence reached 80% in a 6-well cell culture plate, the cells were cultured for 24 h in a basal medium supplemented with 2 μg/mL prolactin (Sigma). Subsequently, these cells were transfected with EGFP-SOCS1 (3 μg), EGFP-CEBPA (3 μg), siCEBPA (60 nM) and the corresponding negative controls (EGFP and siNC) according to the manufacturer’s protocol of TransIntroTM EL transfection reagent (TransGen Biotech, Beijing, China). At the same time, they were transduced into shSOCS1 and shNC to knock down SOCS1. Through pre-experiments, it was determined that the best results were obtained when the cells were collected 48 h after treatment. Therefore, the cells of each treatment group were harvested 48 h later for gene and protein expression analysis.
Buffalo mammary epithelial cells (BuMECs) were isolated from the mammary gland tissue of lactating Binglangjiang buffalo (60 d postpartum) and purified based on the differential sensitivity of the cells to trypsin digestion as previously described by our group [22 (link),23 (link)]. The BuMECs purified and identified by cytokeratin 18 (Sigma, Louis, MO, USA) were cultivated and expanded to passage five in a basal medium composed of DMEM (Gibco), 10% fetal bovine serum (Gibco), 100 μg/mL penicillin/streptomycin (Gibco) and various cytokines, including 5 μg/mL insulin (Sigma), 2 μg/mL hydrocortisone (Sigma) and 100 ng/mL epidermal growth factor (Sigma), and maintained at 37 °C under 5% CO2. When the cell confluence reached 80% in a 6-well cell culture plate, the cells were cultured for 24 h in a basal medium supplemented with 2 μg/mL prolactin (Sigma). Subsequently, these cells were transfected with EGFP-SOCS1 (3 μg), EGFP-CEBPA (3 μg), siCEBPA (60 nM) and the corresponding negative controls (EGFP and siNC) according to the manufacturer’s protocol of TransIntroTM EL transfection reagent (TransGen Biotech, Beijing, China). At the same time, they were transduced into shSOCS1 and shNC to knock down SOCS1. Through pre-experiments, it was determined that the best results were obtained when the cells were collected 48 h after treatment. Therefore, the cells of each treatment group were harvested 48 h later for gene and protein expression analysis.
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