Annexin 5 pi staining kit

Aβ_1-42, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Senegenin (purity>98%) was purchased from Mansite Pharmaceutical Co., LTD. (Chengdu, China). Dulbecco’s modified Eagle medium (DMEM), penicillin, fetal bovine serum (FBS) and streptomycin were purchased from Solarbio (Beijing, China). The Annexin-V/PI staining kit was purchased from KeyGEN (Nanjing, China). 2′, 7′-dichlorofluorescin diacetate (DCFH-DA) probe and Nuclear and Cytoplasmic Protein Extraction Kit were purchased from Beyotime Biotechnology (Shanghai, China). Antibodies against Akt (cat.no.ab18785), P-Akt (cat.no. ab38449), PI3K (cat.no. ab139307), P-PI3K (cat.no. ab182651), Bax (cat.no. ab232479), Bcl-2 (cat.no. ab59348), Nrf2 (cat.no. ab62352) and β-actin (cat.no. ab179467) were from Abcam (Cambridge, United Kingdom). HO-1 (cat.no. GTX101147) was from Gene Tex (Irvine, CA, USA) and Lamin A (cat.no. 201015-3D6) was from Zen-Bio (Chengdu, China).

Cells were starved for 12 h, then treated with or without staurosporine (STS, Selleck, Houston, TX) at 0.2 μM for 24 h. At last cells were harvested and stained with Annexin V/PI Staining Kit (KeyGEN BioTECH, Shanghai, China). Apoptosis was detected by CytoFLEX (Beckman Coulter). Triplicate independent experiments were performed.

The glioblastoma cell lines U87MG, U251 and T98G were obtained from the ATCC (American Type Culture Collection, Virginia, VA, USA) and cultured in the state key laboratory of biotherapy, west china hospital, Sichuan University, China. The cell proliferation and apoptosis assay were measured by using the MTT method and Annexin V/PI staining kit (Keygen, Nanjing, China), respectively. The Western blot (WB) analysis was performed according to the previous reports and manufacturer's protocols. The experimental procedures were described in Supporting Information.