We conducted an analysis of the expression patterns of the TGF-β/Smad signaling pathway, its receptors and the intracellular Smads including Smad2, Smad3, Smad4 and Smad7. We also investigated the protein expression and subcellular localization of some components of Smads in response to the stimulation of TGF-β1 in the NPC cell lines. Materials and methods Cell lines, Everolimus nmr cell culture and treatment The nasopharyngeal carcinoma cell lines (CNE2)

and the immortalized nasopharyngeal epithelial cell line (NP69) were provided by the Biopharmaceutical Research and Development Center (Jinan University, Guangzhou, China), and cultured in Keratinocyte-SFM medium (Gibco, Carlsbad, CA) at 37°C in a humidified atmosphere of 5% CO2. Regarding the treatment of TGF-β1, the cells were plated at 5 × 103 per well in 96-well plate, and cultured in the presence of 10% FBS for 2 days. Then cells were washed and cultured

with serum-free medium overnight, the next day, cells were Rapamycin treated with TGF-β1 at different concentrations in serum-free medium, and then continued to culture for 24 h, 48 h, 72 h, and 96 h, respectively. Selleck PLX3397 Cell growth response To study the dose/time-effect response of CNE2 to TGF-β1, cells were plated at 5 × 103 per well in 96-well plate, and cultured in Keratinocyte-SFM medium for 24 h. Cells were washed and replaced in growth factors-free medium overnight and then treated with 0, 2.5, 5, 7.5, 10 and 12.5 ng/mL TGF-β1 in Keratinocyte-SFM medium. The status of cell growth was determined at 24, 48, 72 and CYTH4 96 h, respectively, using Cell Counting Kit-8 (CCK-8) (Dojindo Laboratories China, Shanghai, China). CCK-8 solution was added into the plated cells at 10 μl/well, 4 h before each treatment and then the 96-well plate was swirled for 15 min. The spectrophotometrical absorbance of each sample was determined at 450 nm. Analysis of TGF-β receptors and Smads by RT-

PCR Cells were seeded at 1.6 × 105 cells per well into 6-well plate and cultured in Keratinocyte-SFM medium with growth factors for 24 h. Cells were washed and replaced with growth factors-free medium overnight, and then TGF-β1 was added (final concentration 10 ng/mL) for 3 h. Total RNA was isolated by using an RNA extraction kit and RNAex reagent (Huashun Biotechnology Co., Ltd., Shanghai, China) according to the manufacturer’s instruction. Reverse transcription of 2 ng of total RNA was performed by using 20 units of AMV reverse transcriptase (BBI); 0.5 ng of oligo (dT) 12-18 primer; 0.5 mM each of dNTP and 20 units of RNase inhibitor in a total volume of 20 μL at 42°C for 60 min. The reaction was terminated by heating the mixture at 70°C for 10 min, and then was chilled on ice. After reverse transcription, PCR amplification was carried out in a volume of 20 μL containing 1× PCR reaction buffer, 0.2 mM dNTPs, 0.