Knockdown of EGFR with siRNA

Transfection with 10 nM siRNA targeting the EGFR (siEGFR, sc-29301, Santa Cruz Biotechnology, Dallas, USA) or control siRNA (scrambled control, Santa Cruz Biotechnology, Dallas, USA) was performed using the siPORT NeoFX Transfection Agent (AM4511, Thermo Fisher Scientific) according to the manufacturer’s overlay transfection instructions. Adjustments of the transfection protocol were made by using 6 µL of siPORT for 1.8 × 10⁵ cells per 6-well plate. A transfection mix containing siRNA and siPORT was incubated 15 min prior to overlay of cells. Knockdown was performed over 48 h and 72 h.