P h2ax

Dimethylsulfoxide (DMSO), 3-(4,5-dimethylthylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), and all other chemicals were purchased form Sigma-Aldrich (St. Louis, MO, USA). Antibodies against PARP, caspase-3, caspase-8, caspase-9, Bak, IRE1α, PERK, TNFR2, CDK6, p-cdc2, p-Rb (ser795), p-ATM, p-ATR, p-Chk1, and p-Chk2 were purchased from Cell Signaling Technologies (Beverly, MA, USA). Antibodies of Bcl-2, LC3, NF-κB, p-H2AX, ATF6, and CDK4 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies of TNFR1, RIPK1, RIPK3, and MLKL were purchased from ABclonal (ABclonal, Wuhan, China). Antibodies of Actin and p62/SQSTM1 were purchased from Sigma (Sigma, CA, USA). Anti-mouse and rabbit IgG peroxidase-conjugated secondary antibody were purchased from Pierce (Rockford, IL, USA). Hybond ECL transfer membrane and ECL Western blotting detection kits were purchased from Ameisham Life Sciences (Amersham, Buckinghamshire, UK). Rhodamine 123 and the carboxy derivative of fluorescein (carboxy-H2DCFDA) were purchased from Molecular Probes and Invitrogen detection technologies (Carlsbad, CA, USA).

Cell Counting Kit-8(CCK-8), methanol, dimethylsulfoxide (DMSO),thiostrepton were obtained from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against FOXM1, Aurora Kinase A,B,CDK-6,-4, PARP, cleaved caspase-8,-3, and β-actin were obtained from Cell Signaling Technologies (Beverly, MA, USA).BUB1B, HSP60, p-H2AX, Bax, Bcl2 were obtained from Santa Cruz Biotechnology, Inc., (CA, USA). AllPrep DNA/RNA Mini Kit was purchased from Qiagen (Hilden, Germany). Live and Dead assay kit, RPMI 1640 medium, fetal bovine serum (FBS), penicillin and streptomycin were obtained from Life Technologies, Inc. (Carlsbad, CA).

Formalin-fixed, paraffin-embedded tissues were cut into 5-μm sections for IHC analysis. Antibodies specific for IL-6, p-H2AX, Ki-67, and active caspase 3 were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), Research & Diagnostics Systems, Inc. (Minneapolis, MN, USA), Cell Signaling Technology (Danvers, MA, USA), and Chemicon (Temecula, CA, USA). The sections were incubated overnight at 4 °C with antibodies against the target proteins. Frozen tissue specimens were cut into 5–8 μm cryostat sections, warmed to room temperature, fixed for 10 min in cold acetone (−20 °C), and incubated for 20 min in PBS containing 10% goat serum. The sections were incubated overnight at 4 °C with antibodies against IL-6, CD11b, and CD3, washed three times with PBS, and incubated for 1 h with fluorescein or Texas Red-conjugated secondary antibodies. The positive staining signals were assessed by microscope from ten random fields and semiquantitated by MetaMorph software (version 7.7, Molecular Devices, Sunnyvale, CA, USA)