HEC-1A and HEC-1B cells were removed from the culture vessels with 0.25 % (w/v) trypsin-EDTA (Gibco, Thermo Fisher, UK) and re-suspended in cell culture media. 1 ml of cell suspension (approximately 8 × 104 cells) was aspirated onto round glass-bottomed 30 mm dishes (WillCo Wells, Amsterdam, The Netherlands), and cells allowed to attach for 5 mins. 2 ml of cell culture media was then added and cells incubated for 24 h (37 °C, 5 % CO2). The cell culture medium was then replaced with 2.5 ml antibiotic free culture media supplemented with 500 µl OPTI-C medium, 10 µl MUC1 siRNA (Santa Cruz Biotechnology, Heidelberg, Germany) and 7.5 µl lipofectamine RNAiMAX (Invitrogen) before the cells were further incubated for 48 h (37 °C, 5 % CO2). This transfection preparation was incubated for 20 mins at 25 °C prior to application.

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