Welfect ex plus reagent

Human SW620 colon cancer cell lines were obtained from the American Type Culture Collection (Manassas, VA). Colon cancer cells were incubated with medium RPMI 1640 containing L-glutamine 1X supplemented with 10% fetal bovine serum (FBS), 1% 10000 U/ml penicillin and 10000 μg/ml streptomycin, at 37°C in 5% CO2 humidified air. All reagents were purchased from Invitrogen (Carlsbad, CA, USA). To establish constitutive expression systems of IL-32α, cancer cells were transfected with the pcDNA3.1 or pcDNA3.1-IL-32α vector using the Lipofectamine™ 2000 (Invitrogen). The transfected cells were selected with an antibiotic for neomycin resistance gene, G418 (800 μg ml, Sigma). G418-resistant cells were screened for 3 weeks, and single cell-expanded clones were obtained by serial dilutions. We confirmed the stable expression of IL32α in SW620 cells using the Western blot. The cells were transiently transfected with TNFR1 siRNA (Santa Cruz Biotechnology) or Control siRNA (Santa Cruz Biotechnology) per well using a mixture of siRNA and WelFect-EX Plus reagent in OPTI-MEM, according to the manufacturer's specification (WelGENE, Seoul, Korea).