Cell culture and transfection

PTC cell lines, TPC-1 and ARO, were obtained from the Chinese Academy of Sciences (Shanghai, People’s Republic of China) and cultured in Dulbecco’s Minimum Essential Medium (DMEM) containing 10% fetal calf serum (FCS) and antibiotics in a humidified atmosphere with 5% CO₂ at 37°C. Cells were digested with trypsin and seeded into a six-well plate (5×10⁵ cells/well) before transfection. Lipofectamine™ 2000 (Invitrogen) was used to transfect IL13Rα2 siRNAs (siRNA-1, GGCUAUCGGAUGCUUAUAU); siRNA-2 (CAGGAUAUGGAUUGCGUAU) or negative control siRNA (NC) into PTC cells. At 48 hours posttransfection, cells were collected and evaluated for knockdown effect by using real-time PCR and Western blot assay. Then, transfected TPC-1 and ARO cells were processed for Cell Counting Kit-8 (CCK-8), flow cytometry, Transwell, biochemistry, Western blot, and real-time PCR analyses.