Zd1839

SiHa cells were purchased from the Chinese Academy of Sciences cell bank (Shanghai, China). Cells were plated in 6-well plates at ~2×10⁶ viable cells per well in 1 ml of Dulbecco's modified Eagle medium (Solarbio Science and Technology Co., Ltd., Beijing, China) containing 10% fetal bovine serum (Zhejiang Tianhang Biotechnology Co., Ltd., Hangzhou, China), and cultured at 37°C in an atmosphere of 5% CO₂ and 95% air. Cells were allowed to attach for 24 h and then incubated in serum-free medium for 16–18 h. Subsequently, the cells were treated with EGF (PeproTech Inc., Rocky Hill, NJ, USA) in the presence or absence of methyl-β-cyclodextrin (MβCD; 0.5 nM; Sigma-Aldrich; Merck Millipore, Darmstadt, Germany), cholesterol (10 µM; Sigma-Aldrich; Merck Millipore) and inhibitors of EGFR (ZD1839; 10 nM; AstraZeneca, London, UK), PI3-K (LY294002; 20 nM; and wortmannin; 5 nM), MAPK kinase (MEK) (PD98059; 20 nM; and U0126; 10 nM), p38 (SB203580; 10 nM) and JNK (SP600125; 10 nM; all Sigma-Aldrich; Merck Millipore) for 1 h prior to exposure to EGF. Cells were harvested after incubation with EGF for the indicated length of time.