Cell culture and Transfection
RAW264.7 cells obtained from Zhong Qiao Xin Zhou Biotechnology were cultured in DMEM supplemented with 10% fetal bovine serum, 100 units/mL penicillin and 100 g/mL streptomycin at 37 C in 5% CO2. siNR4a1 and plasmid were Designed and produced by IGE biotechnology. The sequence of siNR4a1: Forward:GCCAUUAGAUGAGACCUAUdTdT Reverse:AUAGGGUCUCA
UCUAAUGGGCdTdT. siRNA and plasmid Transfection was conducted by lipofectamine 3000 (Thermo Fisher Scientific). After 40 hours transfection, cells were stimulated by LPS for another 8 hours for further testing.