Fgfr1

Control and knockdown ZEB1, FGFR1, FGFR2 and FGFR3 plasmids were purchased from Dharmacon. Plasmids for expression of miR-203 and miR-200 were a gift from Thomas Brabletz (University of Erlangen, Germany). Control and knockdown ADAMDEC1 plasmids were purchased from Sigma. Different clones for each shRNA plasmid were tested and the best knockdowns were selected to produce lentiviral particles. A plasmid for overexpression of FGFR1 was a gift from Dominic Esposito (Addgene plasmid #70367) and cloned into an expression vector (pHIV-IRES-mRFP) using the Gateway system (Invitrogen). Lentiviral particles were generated by co-transfecting HEK293T cells with second generation packaging plasmids (psPAX2 and pMD2.G) using Lipofectamine 3000 (Invitrogen). Medium containing lentiviral particles was collected 48 h and 72 h after transfection. Viral supernatants were combined and filtered with a 0.45 µm pore size filter, followed by ultracentrifugation at 185,000 rcf with a L8–70M Ultracentrifuge (Beckman). Pelleted viral particles were diluted in 200 µl of N2 medium. Concentrated viral particles were aliquoted and stored at −80°C.
For lentiviral transduction, 1×10⁵ cells were pre-incubated for 1–2 h in N2 medium without antibiotics and 1µg/µl of polybrene (Santa Cruz) to increase transduction efficiency. 18h after transduction, medium was replaced with complete N2 medium and growth factors.

Cells were seeded into 6-well plates at a density of 1–2 × 10⁵ cells/well. Twenty-four hours later, cells were transfected with 20 nM siRNA against FGFR1 (Dharmacon), DUSP6 (Dharmacon), ZEB1 (Dharmacon and Santa Cruz Biotechnology), SPRY4 (Ambion and Dharmacon), or ERBB3 (Invitrogen) or Stealth RNAi-negative control low GC Duplex #3 (Invitrogen) using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions.