Journal: PLoS ONE

Article Title: FGFR2 Is Amplified in the NCI-H716 Colorectal Cancer Cell Line and Is Required for Growth and Survival

doi: 10.1371/journal.pone.0098515

Figure Lengend Snippet: A. NCI-H716 cells were treated with colcemid (0.02 µg/ml for 3 hours), fixed with methanol/acetic acid, and dropped onto a microscope slide according to materials and methods. Bacterial artificial chromosome clone RP11-62L18 was labeled with Spectrum Orange dUTP and a centromere probe was labeled with Spectrum Green dUTP (Abbott Molecular Inc., Des Plaines, IL) and hybridized to fixed cells. Red indicates FGFR2 and green indicates Centromere. B. FGFR2 is overexpressed and contains high levels of tyrosine phosphorylation in the NCI-H716 cell line. A, Cell lysates (prepared according to Materials and Methods) from untreated or FGF2 treated (30 ng/ml, 5 minutes) cells were lysed and protein concentration was determined with BCA kit (Pierce Thermo Fisher Rockford Ill). Equal lysate amounts (50 ug) were subjected to SDS-PAGE and western blotting with FGFR2 antibodies made against the N terminus (H80. MAB6841), C terminus (C20) and activation loop phosphorylation Y653/654 (3471, p-FGFR2) and Actin.

Article Snippet: Antibodies against FGFR2 included N-terminus MAB6841 (R&D Systems, Minneapolis, MN) and H80 (Santa Cruz), C-terminus C-20 (Santa Cruz) and Y653/654 specific 3471 ((Cell Signaling Technology, (CST), Danvers MA.)).

Techniques: Microscopy, Labeling, Phospho-proteomics, Protein Concentration, SDS Page, Western Blot, Activation Assay

Journal: PLoS ONE

Article Title: FGFR2 Is Amplified in the NCI-H716 Colorectal Cancer Cell Line and Is Required for Growth and Survival

doi: 10.1371/journal.pone.0098515

Figure Lengend Snippet: A. PD173074 and MK2461 inhibit FGFR2 phosphorylation. Cells were treated for 1 hour with a titration of PD173074 as described in Materials and methods. Lysates were prepared and 50 ug protein was subjected to SDS-PAGE and western blotting with phospho Y653/654 FGFR and MAB6841 total FGFR2 antibody. B. PD173074 and MK2461 inhibit NCI-H716 cell growth. Cell lines were plated at 4000 cells/well and incubated overnight. NCI-H716 cells were treated with a titration of PD173074 as described in Materials and Methods. Cell growth was measured with Vialight reagent, and growth was presented relative to untreated cells. C. PD173074 and MK2461 selectively inhibit growth of NCI-H716 cells. Colon cancer cell lines listed were plated at 4000 cells/well and 24 hours later were treated with a titration of compounds. 4 days later cell growth was measured with vialight and IC50s were calculated from graph pad prism. D. FGFR2 shRNA decreases FGFR2 protein in NCI-H716 cells. FGFR2 shRNA was prepared and NCI-H716 cells were infected as described in methods. Left, FGFR2 expression was analyzed with phospho Y653/654 and total protein with MAB6841. E. Growth was analyzed 5 days post infection with Vialight reagent.

Article Snippet: Antibodies against FGFR2 included N-terminus MAB6841 (R&D Systems, Minneapolis, MN) and H80 (Santa Cruz), C-terminus C-20 (Santa Cruz) and Y653/654 specific 3471 ((Cell Signaling Technology, (CST), Danvers MA.)).

Techniques: Phospho-proteomics, Titration, SDS Page, Western Blot, Incubation, shRNA, Infection, Expressing

Journal: PLoS ONE

Article Title: FGFR2 Is Amplified in the NCI-H716 Colorectal Cancer Cell Line and Is Required for Growth and Survival

doi: 10.1371/journal.pone.0098515

Figure Lengend Snippet: Colon cancer arrays CO702, 802 and CO992 were stained with the H80 FGFR2 antibody (Santa Cruz Biotechnology, Santa Cruz CA) at a 1∶50 dilution on a Ventana Benchmark. Positive controls: NCI-H716, indicates a section from a NCI-H716 xenograft, and 1182 indicates a primary gastric cancer sample harboring FGFR2 amplification. HT29 is a section from an HT29 xenograft. CO802, A2 indicates a positively staining colorectal cancer section, while CO802 D5 indicates a negatively staining section.

Article Snippet: Antibodies against FGFR2 included N-terminus MAB6841 (R&D Systems, Minneapolis, MN) and H80 (Santa Cruz), C-terminus C-20 (Santa Cruz) and Y653/654 specific 3471 ((Cell Signaling Technology, (CST), Danvers MA.)).

Techniques: Staining, Amplification

Journal: British Journal of Cancer

Article Title: Individuality in FGF1 expression significantly influences platinum resistance and progression-free survival in ovarian cancer

doi: 10.1038/bjc.2012.410

Figure Lengend Snippet: Validation of ovarian tumour histology associations. Single-gene Taqman probes were used to compare gene expression in FFPE ovarian tumours ( n =91) as described in Materials and Methods, and tumours subclassified according to histology – serous tumours ( n =58) and nonserous tumours ( n =33, combined mucinous, endometrioid and clear-cell histologies). WT-1 ( A ), FGF1 ( C ) and FGFR2 ( D ) were significantly more highly expressed in serous tumours; increased WT-1 expression in serous tumours was confirmed by immunohistochemical analysis as described in Materials and Methods ( B ). In contrast, KIT ( E ) was significantly overexpressed in nonserous tumours.

Article Snippet: Analysis was restricted to genes ( FGF1 and KIT ) most significantly associated with tumour histology in our fresh-frozen tumour cohort, with the addition of WT-1 (Taqman probe ID Hs01103749_m1), FGFR1 (Taqman probe ID Hs00915142_m1) and FGFR2 (Taqman probe ID Hs01552926_m1).

Techniques: Biomarker Discovery, Gene Expression, Expressing, Immunohistochemical staining