The human glioblastoma cell lines U87 MG, U118 MG, U251 MG, A172, LN-229, and T98G were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). The cells were cultured in DMEM supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA).
The antibodies against NAG-1, Bcl-2, Bax, caspase-3, caspase-8, caspase-9, cytochrome c, p-PI3K(p85 Tyr458)/PI3K(p85), p-Akt(Ser473)/Akt, p-ERK1/2(Thr202/Tyr204)/ERK1/2, p-Smad2(Ser465/467)/Smad2, p-Smad3(Ser423/425)/Smad3, and β-actin were purchased from Cell Signaling Technologies (Beverly, MA, USA). Wortmannin, LY294002, Smad2 siRNA, and Smad3 siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Ac-IETD-FMK and Ac-LEHD-FMK were purchased from KeyGEN Biotech Co., Ltd. (Nanjing, China). Enhanced chemiluminescence (ECL) detection system was purchased from Amersham Life Science (Arlington Heights, Illinois, USA). Human NAG-1 ELISA Kit was obtained from Huamei Biological Company (Wuhan, China). Mitochondrial membrane potential assay kit with JC-1 and protein A/G agarose beads were obtained from Beyotime Institute of Biotechnology (Shanghai, China). X-treme GENE siRNA transfection reagent was purchased from Roche Applied Science.
The antibodies against NAG-1, Bcl-2, Bax, caspase-3, caspase-8, caspase-9, cytochrome c, p-PI3K(p85 Tyr458)/PI3K(p85), p-Akt(Ser473)/Akt, p-ERK1/2(Thr202/Tyr204)/ERK1/2, p-Smad2(Ser465/467)/Smad2, p-Smad3(Ser423/425)/Smad3, and β-actin were purchased from Cell Signaling Technologies (Beverly, MA, USA). Wortmannin, LY294002, Smad2 siRNA, and Smad3 siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Ac-IETD-FMK and Ac-LEHD-FMK were purchased from KeyGEN Biotech Co., Ltd. (Nanjing, China). Enhanced chemiluminescence (ECL) detection system was purchased from Amersham Life Science (Arlington Heights, Illinois, USA). Human NAG-1 ELISA Kit was obtained from Huamei Biological Company (Wuhan, China). Mitochondrial membrane potential assay kit with JC-1 and protein A/G agarose beads were obtained from Beyotime Institute of Biotechnology (Shanghai, China). X-treme GENE siRNA transfection reagent was purchased from Roche Applied Science.