Smac diablo

For the immunoblotting of P53, Bcl-2, Bax, and caspase-3, proteins were extracted from whole cell lysates using RIPA buffer (Cell Signaling) following manufacturer's instructions. For the immunoblotting of Cytochrome c and Smac, cytoplasmic proteins were extracted using NE-PER Nuclear Protein Extraction Kit (Thermo Fisher). The proteins separated by SDS gel were transferred to 0.22 μm PVDF membrane (Bio-Rad) at 15 V for 2 h by using semidry transfer set (CBS Scientific). After blocking the membranes with 5% nonfat dry milk in Tris-buffered saline with Tween (TBST) (150 mM NaCl, 15 mM Tris-HCl (pH 7.5), and 0.1% Tween 20) for about 2 h, proteins were probed with primary antibody against Bcl-2 (Bioworld), caspase-3 (Bioworld), P53 (Bioworld), Bax (Bioworld), Smac/Diablo (Cell Signaling), Cytochrome c (Cell Signaling), or β-actin antibody (Santa Cruz) and then incubated with a secondary antibody conjugated with horseradish peroxidase (Cell Signaling). Membranes were washed by TBST after each antibody probing. Signals were detected by using Super Signal West Pico chemiluminescent substrate (Thermo Scientific). Integrated optical density (IOD) quantification was performed using Gel-Pro Analyzer 4.0.

Furanodienone (⩽98%) and 5-FU were purchased from Shanghai Yuanye Bio-Technology Co., Ltd, (Shanghai, China). A stock solution of Furanodienone at 4 mM was prepared in dimethyl sulfoxide (DMSO; Sigma, St. Louis, MO, USA) and stored at −20 °C, while that of 5-FU was prepared in phosphate-buffered saline (PBS) and stored at 4 °C. Dulbecco’s modified Eagle’s medium (DMEM) and 1 × (PBS, pH 7.4) were obtained from Jinuo Biotechnology (Hangzhou, China). Caspase-3, -8 and -9 Activity Assay Kits, Reactive oxygen species Assay Kit, BCA protein assay Kit and Cell Cycle and Apoptosis Analysis Kits were purchased from Beyotime Biotechnology (Suzhou, China). The NAC was purchased from Sigma Chemical Co. (St. Louis, MO, USA), and inhibitors z-VAD-fmk, z-LEHD-fmk, U0126, SP600125 and SB202190 were afforded by Shanghai Qiangzhi Bio-Technology Co., Ltd, (Shanghai, China). The primary antibodies: p21^Cip1, CDK 4, cyclin D1, CDK 6, CDK 2, cyclin E, caspase-8, -3 and -9, cleaved caspase-8, -3 and -9, cleaved PARP, Bax, Bcl-2, Bcl-xl, cytochrome c, AIF, Smac/DIABLO, survivin, p-p38, p38, p-JNK, JNK, p-ERK, ERK, α-tubulin and GAPDH were afforded by Cell Signaling Technology (Beverly, MA, USA).

Raw DML plants were obtained from the Xinjiang Institute of Materia Medica, Xinjiang, China. These plants were authenticated by Prof. Jiang He of Xinjiang University of Chinese Medicine. Radio-immunoprecipitation assay (RIPA) lysis buffer was obtained from Beyotime Institute of Biotechnology (Beijing, China). 2,3,5-Triphenyl tetrazolium chloride (TTC) was purchased from Sigma (Beijing, China). Malondialdehyde (MDA), lactate dehydrogenase (LDH), superoxide dismutase (SOD), and serum creatinine kinase MB (CK-MB) assay kits were purchased from Jiancheng Bioengineering Institute (Nanjing, China). LY294002 (PI3K inhibitor) was purchased from Abcam (Beijing, China). Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay kit was purchased from Boster Biological Technology co. Itd (Wuhan, China). The primary antibodies against phosphorylation of PI3K (p-PI3K), PI3K, phosphorylation of Akt (p-Akt), Akt, Bcl-2, Bax, caspase-3, cleaved caspase-3, caspase-7, Caspase-9, Smac/Diablo, HrtA2/Omi, XIAP, GAPDH, and β-actin were purchased from Cell Signaling Technology, Inc (CST, USA). Goat anti-rabbit secondary antibodies were purchased from the Zhongshan Company (Beijing, China).

The total proteins of 293T cells were extracted with lysis buffer [20 mM Tris-HCl, 137 mM NaCl, 10% glycerol, 1% Triton X-100, 2 mM EDTA, supplemented with 0.1% PMSF (Beyotime, China)]. The lysates were shaken on ice for 15 min, and the protein concentration was ascertained by BCA assay. The cell lysates were mixed with 2× SDS sample loading buffer and subjected to 10% or 12% (dependent on predicted protein molecular weight) SDS polyacrylamide gel electrophoresis analysis. The proteins were transferred to a PVDF membrane. After blocking with 5% skim milk in TBS-T (0.12 M Tris-base, 1.5 M NaCl, 0.1% Tween-20), the membrane was incubated with primary antibodies, targeting Cleaved-caspase-3, Cleaved-caspase-9, Cleaved-caspas-8, Cleaved-PARP, Bax, Bak, Bid, Smac/Diablo, cIAP-2, Bcl-2, and Rb (all purchased from Cell Signaling Technologies, Danvers, MA, United States), at 4°C overnight. The blots were washed three times in TBST (20 mM pH 7.4 Tris-HCl, 150 mM NaCl, 0.05% Tween-20) and incubated with secondary goat anti-rabbit/mouse horseradish peroxidase-conjugated IgG. The membranes were washed thrice with TBST and the protein was visualized using an ECL chemiluminescent substrate according to the manufacturer’s instructions (Pierce-Thermo Fisher Scientific, Waltham, MA, United States).