File S1 - FGFR2 Is Amplified in the NCI-H716 Colorectal Cancer Cell Line and Is Required for Growth and Survival

Combined file of supporting figures. Figure S1. FGFR2 copy gain and overexpression in H716 cells. A, B. Highly focal copy gain at the FGFR2 locus at MB 122 in the NCI-H716 cell line and comparison to FGFR2 amplified gastric cancer cell line KATOIII by SNP aCGH from the Sanger Wellcome Trust Institute. C. Oncomine (Compendia Bioscience, Ann Arbor, MI, USA) database reveals selective FGFR2 overexpression in NCI-H716 and in FGFR2 amplified KATOIII and SNU16 cell lines. Figure S2: FGF2 does not further activate FGFR2 in H716 or colon cancer cell lines. Lysates were processed as in Figure 1A. “+” indicates addition of 50 ng/ml FGF2 for 15 minutes. Figure S3: PD173074 is highly selective for FGFR1,2,3. 100 nM PD173074 was tested for inhibition of the listed kinases on the Ambit kinase binding platform. The platform measures PD173074 binding but not inhibition of kinase activity. The far left column indicates that FGFR1,2,3 bind strongly to PD173074, while DDR1, MKNK1, FLT4, PIK3CB, and CSF1R bind 10–20 fold less tightly. The kinases in the columns on the right bind poorly. Because DDR1 and DDR2 are highly homologous in their kinase domain, and PD173074 did not bind to DDR2, this suggests that binding to DDR1 may be outside the conserved kinase domain. Figure S4: PD173074 inhibits pFGFR2 and selectively inhibits NCI-H716 growth. A. FGFR2 phosphorylation in Figure 2 was scanned and quantitated using Image Quant software. IC50 values for inhibition of FGFR2 phosphorylation are indicated. B. Tyrosine kinase inhibitors that lack FGFR2 inhibition do not block growth of NCI-H716 cells. Compounds were used at 1 uM (Gleevec, Tarceva, PD168393), 500 nM (Lapatinib, PHA665752), 100 nM (PD173074) or at 10 ug/ml (anti-IGF1R). NCI-H716 were plated at six thousand cells/well in a 96 well plate, treated with compounds, and processed with Vialight reagent 72 hours later. T = 0 indicates the starting cell number, indicating that PD173074 causes a decrease in starting cell number. Figure S5: pRSK S359/363 (ERK phosphorylation site) is inhibited by PD173074. NCI-H716 cells were untreated or treated with 100 nM PD173074 for 2 hours and processed for western blotting as indicated in materials and methods. PhosphoRSK was detected at the ERK phosphorylation site with S359/363 antibody. Figure S6: L-547 and Rapamycin inhibit Akt and S6RP phosphorylation. NCI-H716 cells were treated with 1 uM L-547 or 3 nM Rapamycin for 4 hours. Cell lysates were processed for western analysis according to Materials and Methods. Figure S7 PD173074 causes cell death in NCI-H716 cells. Cells were treated for 72 hours and photographed. Fragmented cells are consistent with cell death after PD13074 treatment. Figure S8: E cadherin and EPCAM are not expressed in H716 cells. Western lysates from Figure 1B were blotted for adhesion molecules E-Cadherin (CDH1) and EPCAM. Arrows indicate NCI-H716 and RKO cells that do not express of CDH1 and EPCAM. (PPT)