Rage neutralizing antibody

The human prostate cancer PC3 cell line was obtained from the Type Culture Collection of the Chinese Academy of Science (Shanghai, China), and was cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (both Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) in a humidified 5% CO₂ incubator at 37°C. Briefly, PC3 cells at 60–80% confluency were stimulated with 0.5, 1 or 2 µg/ml recombinant (r) HMGB1 (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 24 h at 37°C. For the RAGE inhibition experiment, RAGE neutralizing antibody (R&D Systems, Inc., Minneapolis, MN, USA) was used at 20 µg/ml for 24 h at 37°C.