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, 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: Titration, SDS Page, Western Blot, Incubation, shRNA, Infection, Expressing

Journal: PLoS ONE

Article Title: Mannose Phosphate Isomerase Regulates Fibroblast Growth Factor Receptor Family Signaling and Glioma Radiosensitivity

doi: 10.1371/journal.pone.0110345

Figure Lengend Snippet: A. RTK phospho-array analysis of FGF-1 (50 ng/mL) stimulated U-251 control or MPI- shRNA knockdown cell lines was performed as described previously. Tyrosine phosphorylated (1) FGFR1 (2) FGFR2 and (3) FGFR3 are indicated on the left. Signal intensity was analyzed using Image J software to compare relative levels of FGFR phosphorylation between the cell lines (right panel). B. MPI knockdown blocks FGF-1 induced FGFR signaling. Western blot analysis of control and MPI-shRNA knockdown cell lines stimulated with 50 ng/mL FGF-1 was performed to determine induction of FGFR1, FRS2, Akt, and ERK phosphorylation. Actin expression was used as a control for protein loading. Quantification of Akt and ERK phosphorylation relative to total protein was determined as in 3A and are representative of three experiments (right panel). C. MPI knockdown does not block EGF or HGF/SF RTK signaling. Western blot analysis of control and MPI-shRNA knockdown cell lines stimulated with 10 ng/mL EGF or 30 ng/mL HGF/SF were performed to determine induction of EGFR, Met, Akt, and ERK phosphorylation.

Article Snippet: Mouse monoclonal antibody anti-phosphotyrosine (P-Tyr-100), anti-β-actin (8H10D10), anti-Akt, anti-phosphorylated Akt Ser473 (D9E), anti-phosphorylated mitogen-activated protein kinase antibody, anti-phosphorylated-p44/42 MAPK (Erk1/2), anti-phosphorylated EGFR (Tyr1173), anti-Met, anti-phosphorylated Met (Tyr1234/1235), anti-phosphorylated FGFR (Tyr653/654), anti-phosphorylated HERB3/ErbB3 (Tyr1289), anti-phosphorylated-FRS2-α (Tyr196), were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: shRNA, Software, Western Blot, Expressing, Blocking Assay

Journal: Cell Death & Disease

Article Title: Inhibition of autophagy potentiates the cytotoxicity of the irreversible FGFR1-4 inhibitor FIIN-2 on lung adenocarcinoma

doi: 10.1038/s41419-022-05201-0

Figure Lengend Snippet: a The mRNA levels of FGFR1-4 in A549 and A549/DDP cells were detected by RT-qPCR. b Representative immunohistochemistry images of lung adenocarcinoma (LUAD) tissues with high or low expression of FGFR2. The same tissue was stained with haematoxylin and eosin (H&E) (scale bar: 60 μm, 200×). c The expression of FGFR2 and FGFR-dependent signalling proteins in A549 and A549/DDP cells. d The effects of FIIN-2 on FGFR2 and FGFR-dependent signalling proteins in A549 and A549/DDP cells were detected by western blot analysis. GAPDH and β-tubulin were used as the loading controls. Each bar corresponds to the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001; # p < 0.05, ## p < 0.01, ### p < 0.001.

Article Snippet: The primary antibodies used in this study were phospho-FGFR (p-FGFR, #3471), p-FRS2 α(#3861), p-ERK1/2 (#4370), β-actin (#3700), and GAPDH (#5174 S), and they were purchased from Cell Signalling Technologies (Danvers, MA, USA). β-tubulin (#BSM-33034) was purchased from Bioss Technology Co., Ltd. (Beijing, China).

Techniques: Quantitative RT-PCR, Immunohistochemistry, Expressing, Staining, Western Blot