2.5. P2Y12 Knockdown

AsPC-1 cells were seeded at 5 × 10⁴ cells per well in a 12-well plate and maintained for 24 h. P2Y12 siRNA (Hs_P2RY12_4 FlexiTube Predesigned siRNA directed against human P2RY12, ({"type":"entrez-nucleotide","attrs":{"text":"NM_022788","term_id":"1780222564","term_text":"NM_022788"}}NM_022788, {"type":"entrez-nucleotide","attrs":{"text":"NM_176876","term_id":"1677500914","term_text":"NM_176876"}}NM_176876)) was obtained from Qiagen (Hilden, Germany). Silencer^® negative control (sequence: proprietary, catalogue #AM4635) was obtained from Ambion^® (ThermoFisher). P2Y12 and Silencer siRNA (25 nM) were prepared using a DharmaFECT1 transfection agent (1 µL per well) (Dharmacon, Lafayette CO, USA) in serum/antibiotic-free media. The transfection mixture was then added to the adherent cells in serum-free media. For BxPC-3, cells were seeded at 1.5 × 10⁵ cells per well in complete media in a 6-well plate. P2Y12 siRNA and Silencer (50 nM) were prepared using a DharmaFECT1 transfection agent (1 µL per well) in serum/antibiotic-free media. The transfection mixture was then added and the plate was incubated. After 24 h, the media were then replaced with complete media and incubated for 48 h. Cells were washed in PBS and then lysed with radioimmunoprecipitation assay buffer (RIPA) containing protease inhibitors).