Erk1 2 inhibitor u0126

4-Hydoxy-2-nonenal was bought from Cayman Chemical Co. (Ann Arbor, MI). N-Acetylcysteine was purchased from Sigma Chemical Co. (St. Louis, MO). DMEM/F12, DMEM and fetal bovine serum (FBS) were obtained from GIBCO BRL (Grand Island, NY). DCFH-DA was purchased from Beyotime Institute of Biotechnology (Haimen, China). Antibodies against PARP, MKP-1, Nrf2, p53, Bax, Bcl-xL/S, GAPDH, histone H3 and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against caspase-3, cleaved-caspase-3, total and phospho (p)-ERK1/2 (Thr202/Tyr204), JNK, p-JNK, p38 MAPK, p-p38 MAPK, eIF2α, p-eIF2α (Ser51), LC3A/B and PP2A were products of Cell Signaling Technology (Beverly, MA). Peroxidase-conjugated goat anti-rabbit and goat anti-mouse secondary antibodies were purchased from Huaxingbio Biotechnology Co. (Beijing, China). The annexin V-FITC&PI kit was from Jiamay Biotechnology (Beijing, China). Kinases inhibitors, including ERK1/2 inhibitor U0126, JNK inhibitor SP600125, p38 MAPK inhibitor SB202190, and protein degradation inhibitor MG132 were obtained from Cell Signaling Technology (Beverly, MA). PERK inhibitor GSK2606414 was from Selleck Chemicals (Houston, TX). Other reagents used in this study were ordered from Sigma, unless otherwise stated.

The isolated NK cells were pretreated with pretreated with 10 µmol/L of p38 inhibitor SB203580 (Cell Signaling Technology, Danvers, Massachusetts), 10 µmol/L of ERK1/2 inhibitor U0126 (Cell Signaling Technology, Danvers, Massachusetts) or 20 µmol/L of JNK1/2 inhibitor SP600125 (Calibiochem, San Diego, California, USA) for 2 h at 37°C. The NK cells in the non-blockade control groups were only pretreated with DMSO for 2 h at 37°C. After blockade, the NK cells were ready for HMGB1 stimulation.

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.