Pcdna3

The cell line used for lentivirus vector transfection in the experiment was the RLE-6TN cell line (The Cell Bank of Xiangya Medical College; ACE II cell line from rats). This cell line was grown in M199 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Hyclone; SH30070.03), penicillin (10,000 U/ml) and streptomycin (10,000 U/ml) (Hyclone; SV 30010). Cells were cultured in an incubator at 37°C and 5% CO₂. The cells in the control group were not manipulated. The cells in short-hairpin (sh)-negative control (NC) group were infected using a negative control viral plasmid, while the cells in sh-IKKβ group were infected by IKKβ shRNA interference (20 µl of virus solution per well) (RNAi) virus. Cells in the NC group were infected by empty pcDNA3.1 virus (Hunan Fenghui Biotechnology Co., Ltd; 0 µl of virus solution per well) and cells in the IKKβ group were infected with the pcDNA3.1-IKKβ overexpression virus. Cells except the control group were all stimulated with LPS at a concentration of 50 µg/ml for 24 h.

A plasmid used for overexpressing circBRWD1 and an empty control plasmid (pcDNA3.1) were purchased from Hunan Fenghui Biotechnology, China, and plasmid sequences were verified via sequencing. Transformed Escherichia coli was added to Luria-Bertani medium (LB) medium (5 g/L yeast powder [Solarbio, Y8020], 10 g/L sodium chloride [Aladdin, C111533], and 5 g/L tryptone [Solarbio, T8490]) and incubated on a shaker for 14 h. Plasmid extraction was performed using a Plasmid Midi Kit (QIAGEN, 12145) according to manufacturer instructions.