Cell cultures and reagents
Non-small
cell lung cancer cell line A549(Shanghai Institute of Cell Biology,
China) was cultured in RPMI medium 1640 (Life Technologies Corporation,
Grand Island, NY, USA)added with 10 % fetal bovine serum (Gibco
Corporation, Carlsbad, CA,USA), 1 mM glutamine 1.5 g/l sodium
bicarbonate, and 1 mM sodium pyruvate at5 % CO 2. 37
°C. The Antibodies against Bcl-2,Bax,PARP-1,Mcl-1, Bcl-xL, Bcl-2,
Bax,caspase-3,caspase-8, caspase-9, PARP, AMPK, p-AMPK,,Akt,
p-Akt,,c-Jun,,p-c-Jun,,JNK,,ULK1,p-JNK,,mTOR,Beclin-1,LC3, p62 and
GAPDH, DAPI, Tom20 were purchased from Cell Signaling Technology.
cyclinA, cyclin B2,cyclin D1,cyclin D3, p21and Cdc2were purchased from
Sigma-Aldrich. Analytical grade NCTD was obtained from Sigma-Aldrich
(St. Louis, MO, USA). A stock solution of NCTD (50 mM) made in
Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Victoria,
Australia) was prepared and stored at -20 ° C. As described previously
[8], the 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazole
bromide (MTT) method was used to detect the drug on cells Proliferation
inhibition rate