Sapk jnk inhibitor sp600125

Human cervical cancer epithelial (HeLa229) cells obtained from the American Type Culture Collection were grown in 6-well, 24-well, or 96-well flat-bottom plates (Corning incorporated, Corning, NY, USA) to a suitable density, and maintained in Dulbecco’s modified minimal essential medium (DMEM; Hyclone, USA) supplemented with 10% fetal bovine serum (Gibico, NY, MA). After culturing overnight at 37°C in an incubator supplied with 5% CO₂, the cell culture medium was replaced with serum-free DMEM. For individual experiments with or without 50 μM ERK1/2 inhibitor U0126 (Cell Signaling Technology, Beverly, MA, USA) or 30 μM SAPK/JNK inhibitor SP600125 (Sigma-Aldrich, Munich, Germany), recombinant CPSIT_0846 was added to the culture medium at different concentrations for different times. For induction of apoptosis, after a 20 h treatment with CPSIT_0846, 1 μM staurosporine (STS; Sigma-Aldrich) was added over a period of 4 h, or 10 μM carbonyl cyanide 3-chlorophenylhydrazone (CCCP; Sigma) was added for 30 min.