siRNA validation of FGFR knockdown

Cell lines were transfected with 50nmol/L of siRNA using Lipofectamine RNAi MAX (Invitrogen) according to manufacturer’s instructions. siRNA sequences are shown in Supplemental Table 5. The day after transfection (day 2), cells were seeded for the viability assays or RNA extraction. On day 3, cells were treated with gefitinib or vehicle. Cell viability was determined after 72 hours of drug treatment using CellTiter-Glo viability assay (Promega) according to manufacturer’s instructions. RNA was extracted after 24 hours of drug treatment using the RNeasy Kit (Qiagen). cDNA was prepared from 500 ng total RNA with First Strand Synthesis Kit (Invitrogen) using oligo-DT primers and quantitative PCR was performed using FastStart SYBR Green (Roche) on a Lightcycler 480. mRNA expression relative to the mRNA levels of the housekeeping gene β-actin was calculated using the delta-delta threshold cycle (∆∆CT) method. Primer sequences are listed in Supplemental Table 4. Relative gene expression levels were determined at baseline (for FGFR1) or after gefitinib treatment (for FGFR2 and FGFR3, which were expressed at very low levels at baseline).