Sineg

CSF-1-specific small interfering (si)RNA (siCSF-1; sense, 5′-ACGUGGCUAAAGUGUUAAAG-3′; antisense, 5′-CCUGUUCUGCAGUUCCUUCCUUGU-3′), and its corresponding non-silencing negative control siRNA (siNeg; sense, 5′-GGCAAAUUGCCCUUAUCCA-3′; antisense, 5′-AACGUUUAAACCGGUUACGUA3′) were designed and synthesized by GeneChem, Inc. (Shanghai, China). ECC-1 or HEC-1A cells (50% seeding density) were cultured in DMEM supplemented with 10% heat-inactivated fetal calf serum (Thermo Fisher Scientific, Inc.) at 37°C under 5% CO₂ in a humidified incubator. Cells in the exponential growth phase were grown for 24 h and then transfected using Lipofectamine^® 3000 (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The concentration of siCSF-1 and siNeg were maintained at 100 nM. The culture medium was replaced with DMEM plus 10% FBS after 6 h of transfection, then 10 µg/ml of puromycin was added at 37°C for 24 h for screening positive cells, and 2 ug/ml puromycin were added to the cell culture medium to maintain positive cell selection. ECC-1 or HEC-1A cells transfected with siCSF-1 and siNeg were used for wound healing assay and Chemotactic migration assay.