Journal: Frontiers in Genetics

Article Title: CircARVCF Contributes to Cisplatin Resistance in Gastric Cancer by Altering miR-1205 and FGFR1

doi: 10.3389/fgene.2021.767590

Figure Lengend Snippet: Primers sequences used for qRT-PCR.

Article Snippet: Next, the slides were incubated with anti-FGFR1 (bs-0230R; Bioss) overnight and secondary antibody (bs-0293G-HRP) for 1 h. After that, the signal was developed with DAB solution and counterstained with hematoxylin (Sigma-Aldrich) as previously described ( ).

Techniques:

Journal: Frontiers in Genetics

Article Title: CircARVCF Contributes to Cisplatin Resistance in Gastric Cancer by Altering miR-1205 and FGFR1

doi: 10.3389/fgene.2021.767590

Figure Lengend Snippet: CircARVCF regulated FGFR1 expression by targeting miR-1205. (A) The complementary sequences between FGFR1 and miR-1205 were exhibited. (B,C) The relationship between FGFR1 and miR-1205 was analyzed by dual-luciferase reporter assay. (D,E) The mRNA and protein levels of FGFR1 in GC tissues and normal tissues were measured by qRT-PCR or western blot assay. (F) The protein level of FGFR1 in GES-1, MKN-45 and AGS cells was measured via western blot assay. (G,H) The mRNA and protein levels of FGFR1 in DDP-resistant and DDP-sensitive GC tissues were quantified by qRT-PCR or western blot. (I) The protein level of FGFR1 in MKN-45, MKN-45/DDP, AGS and AGS/DDP cells was measured via western blot assay. (J) The protein level of FGFR1 in MKN-45/DDP and AGS/DDP cells transfected with miR-NC, miR-1205, miR-1205 + pcDNA or miR-1205 + FGFR1 was measured via western blot assay. (K) The protein level of FGFR1 in MKN-45/DDP and AGS/DDP cells transfected with si-NC, si-circARVCF#1, si-circARVCF#1+in-miR-NC or si-circARVCF#1+in-miE-1205 was measured through western blot assay. * p < 0.05.

Article Snippet: Next, the slides were incubated with anti-FGFR1 (bs-0230R; Bioss) overnight and secondary antibody (bs-0293G-HRP) for 1 h. After that, the signal was developed with DAB solution and counterstained with hematoxylin (Sigma-Aldrich) as previously described ( ).

Techniques: Expressing, Luciferase, Reporter Assay, Quantitative RT-PCR, Western Blot, Transfection

Journal: Frontiers in Genetics

Article Title: CircARVCF Contributes to Cisplatin Resistance in Gastric Cancer by Altering miR-1205 and FGFR1

doi: 10.3389/fgene.2021.767590

Figure Lengend Snippet: MiR-1205 regulated the DDP resistance and malignant behaviors of DDP-resistant GC cells by targeting FGFR1. MKN-45/DDP and AGS/DDP cells were transfected with miR-NC, miR-1205, miR-1205 + pcDNA or miR-1205 + FGFR1. (A,B) DDP resistance was analyzed by CCK-8 assay. (C–F) The colony formation, migration, invasion and apoptosis of MKN-45/DDP and AGS/DDP cells were evaluated by colony formation assay, transwell assay and flow cytometry analysis, respectively. (G,H) The protein levels of MRP1, MDR1, Bcl-2 and Bax in MKN-45/DDP and AGS/DDP cells were measured via western blot assay. * p < 0.05.

Article Snippet: Next, the slides were incubated with anti-FGFR1 (bs-0230R; Bioss) overnight and secondary antibody (bs-0293G-HRP) for 1 h. After that, the signal was developed with DAB solution and counterstained with hematoxylin (Sigma-Aldrich) as previously described ( ).

Techniques: Transfection, CCK-8 Assay, Migration, Colony Assay, Transwell Assay, Flow Cytometry, Western Blot

Journal: Frontiers in Genetics

Article Title: CircARVCF Contributes to Cisplatin Resistance in Gastric Cancer by Altering miR-1205 and FGFR1

doi: 10.3389/fgene.2021.767590

Figure Lengend Snippet: CircARVCF enhanced DDP resistance in GC in vivo . (A,B) Tumor volume and tumor weight were examined. (C,D) The levels of circARVCF and miR-1205 in the tumors were examined by qRT-PCR. (E,F) The level of FGFR1 in the tumors was examined by western blot assay and IHC assay. * p < 0.05.

Article Snippet: Next, the slides were incubated with anti-FGFR1 (bs-0230R; Bioss) overnight and secondary antibody (bs-0293G-HRP) for 1 h. After that, the signal was developed with DAB solution and counterstained with hematoxylin (Sigma-Aldrich) as previously described ( ).

Techniques: In Vivo, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Genetics

Article Title: CircARVCF Contributes to Cisplatin Resistance in Gastric Cancer by Altering miR-1205 and FGFR1

doi: 10.3389/fgene.2021.767590

Figure Lengend Snippet: The frame diagram of circARVCF/miR-1205/FGFR1 axis in regulating DDP resistance and cell progression in GC.

Article Snippet: Next, the slides were incubated with anti-FGFR1 (bs-0230R; Bioss) overnight and secondary antibody (bs-0293G-HRP) for 1 h. After that, the signal was developed with DAB solution and counterstained with hematoxylin (Sigma-Aldrich) as previously described ( ).

Techniques:

Journal: Cells

Article Title: Targeted PARP Inhibition Combined with FGFR1 Blockade is Synthetically Lethal to Malignant Cells in Patients with Pancreatic Cancer

doi: 10.3390/cells9040911

Figure Lengend Snippet: Enhanced FGFR1 and PARP1 expression characterizes FGFR1 inhibitor-resistant pancreatic cells at mRNA and protein levels. ( A ) Histograms of the FGFR1 (upper panel) and PARP1 (lower panel) RNAseq expression profile in dasatinib-sensitive PANC0403, PANC0504, and PANC1005 cells compared with dasatinib-resistant SU8686, MiaPaCa2, and PANC-1 pancreatic cancer cells in the GSE9357/GP10558/GDS5627 cohort. ( B ) Representative Western blot data showing the differential expression of FGFR1 and PARP in PANC-1, AsPC1, PANC0403, or SUIT-2 cells. ( C ) Line graphs showing the effect of 1–10 μM PD173074 or olaparib on the viability of PANC-1 (left panel) or SUIT-2 (right panel) cells. ( D ) Representative Western blot images of the effect of 4.5 μM PD173074 or 3.5 μM olaparib on the expression level of PARP or FGFR1 protein in the PANC-1 and SUIT-2 cells. β-actin served as the loading control.

Article Snippet: Next, the PVDF membranes were blocked in 5% nonfat milk in Tris-buffered saline with Tween 20 for 1 h. After blocking, the membranes were incubated at 4 °C overnight with primary antibodies against FGFR1 (#9740; 1:1000, Cell Signaling Technology Inc., Danvers, MA, USA), PARP (#9532; 1:1000, Cell Signaling Technology), cleaved PARP (#5625; 1:1000, Cell Signaling Technology), caspase-9 (#9508; 1:1000, Cell Signaling Technology), caspase-3 (#9668; 1:1000, Cell Signaling Technology), Bcl-xL (#2764; 1:1000, Cell Signaling Technology), and β-actin (sc-69879; 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Expressing, Western Blot, Quantitative Proteomics, Control

Journal: Cells

Article Title: Targeted PARP Inhibition Combined with FGFR1 Blockade is Synthetically Lethal to Malignant Cells in Patients with Pancreatic Cancer

doi: 10.3390/cells9040911

Figure Lengend Snippet: Aberrant FGFR1 and PARP1 expression is associated with cancer stem cell-like phenotype, regulates DNA repair, and modulates response to therapy in PDAC cells. ( A ) Oncoprint analysis of FGFR1, PARP1, ALDH1A1, ABCB1, RAD51, and H2AFX genetic alterations in the provisional TCGA pancreatic adenocarcinoma cohort. ( B ) Expression profile and ( C ) multidimensional scaling correlation matrix with ( D ) k-means clustering of FGFR1, PARP1, ALDH1A1, ABCB1, RAD51, and H2AFX mRNA in the provisional TCGA pancreatic adenocarcinoma cohort. ( E ) Gene-set expression-based cohort stratification into therapy responders and non-responders using the E-GEOD-17891, AFFY_HG_U133_PLUS_2 dataset ( n = 47). Clustering was based on correlation distance and average linkage. Unit variance scaling is applied to rows; Singular-value decomposition with imputation was used for principal component (PC) calculation. X and Y axes show PC 1 and PC 2, which explain 43.3% and 18.2% of the total variance, respectively. Missense (VUS): Missense variants of uncertain significance

Article Snippet: Next, the PVDF membranes were blocked in 5% nonfat milk in Tris-buffered saline with Tween 20 for 1 h. After blocking, the membranes were incubated at 4 °C overnight with primary antibodies against FGFR1 (#9740; 1:1000, Cell Signaling Technology Inc., Danvers, MA, USA), PARP (#9532; 1:1000, Cell Signaling Technology), cleaved PARP (#5625; 1:1000, Cell Signaling Technology), caspase-9 (#9508; 1:1000, Cell Signaling Technology), caspase-3 (#9668; 1:1000, Cell Signaling Technology), Bcl-xL (#2764; 1:1000, Cell Signaling Technology), and β-actin (sc-69879; 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Expressing

Journal: Cells

Article Title: Targeted PARP Inhibition Combined with FGFR1 Blockade is Synthetically Lethal to Malignant Cells in Patients with Pancreatic Cancer

doi: 10.3390/cells9040911

Figure Lengend Snippet: FGFR1 and PARP1 co-occur and directly interact with each other at the expense of RAD51/H2AFX signaling and with adverse prognostic implications. ( A ) Co-occurrence and mutual exclusivity analysis of FGFR1, PARP1, ALDH1A1, ABCB1, RAD51, and H2AFX in the TCGA pancreatic adenocarcinoma cohort. ( B ) FGFR1-PARP1 gene expression correlation in the TCGA pancreatic adenocarcinoma cohort. ( C ) Schrödinger’s PyMOL molecular graphics system-generated molecular docking of PARP1 and FGFR1. ( D ) PANC-1 and SUIT-2 cells were subjected to immunoprecipitation (IP) with FGFR1 (upper) or PARP1 (lower) antibody followed by Western blotting analysis with the indicated antibodies, with IgG serving as the control. ( E ) Interaction of PARP1 with fibroblast growth factor receptor 1 (FGFR1). Coimmunolocalization assays of PARP1 and FGFR1 in the PANC1 cancer cell lines expressing wild-type PARP1 and FGFR1. Kaplan–Meier plots showing the differential overall survival (OS) between ( F ) FGFR1-low and FGFR1-high samples or ( G ) PARP1-low and PARP1-high samples from the TCGA pancreatic adenocarcinoma cohort.

Article Snippet: Next, the PVDF membranes were blocked in 5% nonfat milk in Tris-buffered saline with Tween 20 for 1 h. After blocking, the membranes were incubated at 4 °C overnight with primary antibodies against FGFR1 (#9740; 1:1000, Cell Signaling Technology Inc., Danvers, MA, USA), PARP (#9532; 1:1000, Cell Signaling Technology), cleaved PARP (#5625; 1:1000, Cell Signaling Technology), caspase-9 (#9508; 1:1000, Cell Signaling Technology), caspase-3 (#9668; 1:1000, Cell Signaling Technology), Bcl-xL (#2764; 1:1000, Cell Signaling Technology), and β-actin (sc-69879; 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Gene Expression, Generated, Immunoprecipitation, Western Blot, Control, Expressing

Journal: Cells

Article Title: Targeted PARP Inhibition Combined with FGFR1 Blockade is Synthetically Lethal to Malignant Cells in Patients with Pancreatic Cancer

doi: 10.3390/cells9040911

Figure Lengend Snippet: Olaparib alone or in synergism with PD173074 suppresses the oncogenic cancer stem cell-like phenotype of PDAC cells through an apoptosis-related impairment of DNA repair. ( A ) Tumorsphere formation assay images and histograms showing the effect of PD173074 and/or olaparib on the tumorsphere formation efficiency of the PANC-1 or SUIT-2 cells. ( B ) Representative colony formation assay data (left) and graph (right) showing the effect of PD17374 and/or olaparib on the number of colonies formed by PANC-1 or SUIT-2 cells. ( C ) Immunofluorescence images and histograms of γ-H2AX (red), RAD51 (green), and nuclear DAPI (blue) staining in PANC-1 cells treated with PD173074 and/or olaparib compared with the control. Scale bar, 10 μm. ( D ) Downregulation of FGFR1, Sox2, and Nanog protein expression levels in PD173074-treated Panc-1 cells as determined by Western blot analysis. β-actin was used as a loading control. * p < 0.05

Article Snippet: Next, the PVDF membranes were blocked in 5% nonfat milk in Tris-buffered saline with Tween 20 for 1 h. After blocking, the membranes were incubated at 4 °C overnight with primary antibodies against FGFR1 (#9740; 1:1000, Cell Signaling Technology Inc., Danvers, MA, USA), PARP (#9532; 1:1000, Cell Signaling Technology), cleaved PARP (#5625; 1:1000, Cell Signaling Technology), caspase-9 (#9508; 1:1000, Cell Signaling Technology), caspase-3 (#9668; 1:1000, Cell Signaling Technology), Bcl-xL (#2764; 1:1000, Cell Signaling Technology), and β-actin (sc-69879; 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Tube Formation Assay, Colony Assay, Immunofluorescence, Staining, Control, Expressing, Western Blot

Journal: Cells

Article Title: Targeted PARP Inhibition Combined with FGFR1 Blockade is Synthetically Lethal to Malignant Cells in Patients with Pancreatic Cancer

doi: 10.3390/cells9040911

Figure Lengend Snippet: In vivo, PDAC cancer stem cells are more sensitive to PD173074 in the absence of PARP1. ( A ) Experimental chart of in vivo studies. Line graphs of the differential effect of ( B ) early or ( C ) late initiation of treatment with PD173074 and/or olaparib on BALB/c mice bearing PANC-1 tumorsphere-derived tumors. Graphical representation of tumor weight at the end of the experiment for ( D ) early and ( E ) late treatment initiation groups. ( F ) Frequency of tumor growth and correlative statistical analysis of treatment regimen in the PD173074- and/or olaparib-treated mice. ( G ) Representative immunohistochemical staining images showing differential expression of cleaved PARP, Ki-67, FGFR1, and Bcl-2 in tumors extracted from the PD173074- and/or olaparib-treated NOD/SCID mice. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Next, the PVDF membranes were blocked in 5% nonfat milk in Tris-buffered saline with Tween 20 for 1 h. After blocking, the membranes were incubated at 4 °C overnight with primary antibodies against FGFR1 (#9740; 1:1000, Cell Signaling Technology Inc., Danvers, MA, USA), PARP (#9532; 1:1000, Cell Signaling Technology), cleaved PARP (#5625; 1:1000, Cell Signaling Technology), caspase-9 (#9508; 1:1000, Cell Signaling Technology), caspase-3 (#9668; 1:1000, Cell Signaling Technology), Bcl-xL (#2764; 1:1000, Cell Signaling Technology), and β-actin (sc-69879; 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: In Vivo, Derivative Assay, Immunohistochemical staining, Staining, Quantitative Proteomics

Journal: Cells

Article Title: Targeted PARP Inhibition Combined with FGFR1 Blockade is Synthetically Lethal to Malignant Cells in Patients with Pancreatic Cancer

doi: 10.3390/cells9040911

Figure Lengend Snippet: Schematic abstract showing targeted inhibition of PARP1 combined with FGFR1 blockade concomitantly suppresses DNA repair and inhibits the viability, colony formation, and tumorsphere formation of resistant pancreatic cells. Furthermore, suppressed tumor growth in vivo indicates the synthetic lethality of the dual PARP1/FGFR1 inhibition to malignant cells in patients with pancreatic cancer.

Article Snippet: Next, the PVDF membranes were blocked in 5% nonfat milk in Tris-buffered saline with Tween 20 for 1 h. After blocking, the membranes were incubated at 4 °C overnight with primary antibodies against FGFR1 (#9740; 1:1000, Cell Signaling Technology Inc., Danvers, MA, USA), PARP (#9532; 1:1000, Cell Signaling Technology), cleaved PARP (#5625; 1:1000, Cell Signaling Technology), caspase-9 (#9508; 1:1000, Cell Signaling Technology), caspase-3 (#9668; 1:1000, Cell Signaling Technology), Bcl-xL (#2764; 1:1000, Cell Signaling Technology), and β-actin (sc-69879; 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Inhibition, In Vivo

Journal: PLoS ONE

Article Title: A Functional Role of Fibroblast Growth Factor Receptor 1 (FGFR1) in the Suppression of Influenza A Virus Replication

doi: 10.1371/journal.pone.0124651

Figure Lengend Snippet: Primer pairs used for real-time PCR.

Article Snippet: Membranes with total FGFR proteins were blocked by 5% (w/v) non-fat dry milk (GE Healthcare), while those with phosphorylated FGFR1 protein were treated with 5% (w/v) bovine serum albumin (BSA) for 1 h at room temperature and then incubated overnight at 4°C with primary antibodies including anti-FGFR1 polyclonal antibody (1:500 dilution, Cell Signaling Technology), anti-phosphorylated FGFR1 polyclonal antibody (1:1000 dilution, Lianke Biotechnology Ltd.), anti-FGFR4 monoclonal antibody (1:1000 dilution, Abcam; Epitomics), and anti-β-actin monoclonal antibody (1:5000 dilution, Sigma-Aldrich).

Techniques:

Journal: PLoS ONE

Article Title: A Functional Role of Fibroblast Growth Factor Receptor 1 (FGFR1) in the Suppression of Influenza A Virus Replication

doi: 10.1371/journal.pone.0124651

Figure Lengend Snippet: ( A - D ) A549 cells were infected with PR8 virus at an MOI of 1 for the indicated times. FGFR mRNA levels were detected using real-time PCR analysis. The mRNA expression of FGFR1 ( A ), FGFR2 ( B ), FGFR3 ( C ), and FGFR4 ( D ) relative to the reference gene GAPDH was calculated. ( E , F ) The lysates of A549 cells were obtained at the indicated times post-PR8 infection. Protein levels of FGFR1 ( E ) and FGFR4 ( F ) were determined by Western blotting using specific antibodies. Densitometric analysis relative to β-actin levels was expressed as fold change. All graphs present the means ± s.e.m. (n = 3). Values of P<0.001 *** were considered statistically highly significant.

Article Snippet: Membranes with total FGFR proteins were blocked by 5% (w/v) non-fat dry milk (GE Healthcare), while those with phosphorylated FGFR1 protein were treated with 5% (w/v) bovine serum albumin (BSA) for 1 h at room temperature and then incubated overnight at 4°C with primary antibodies including anti-FGFR1 polyclonal antibody (1:500 dilution, Cell Signaling Technology), anti-phosphorylated FGFR1 polyclonal antibody (1:1000 dilution, Lianke Biotechnology Ltd.), anti-FGFR4 monoclonal antibody (1:1000 dilution, Abcam; Epitomics), and anti-β-actin monoclonal antibody (1:5000 dilution, Sigma-Aldrich).

Techniques: Infection, Virus, Real-time Polymerase Chain Reaction, Expressing, Western Blot

Journal: PLoS ONE

Article Title: A Functional Role of Fibroblast Growth Factor Receptor 1 (FGFR1) in the Suppression of Influenza A Virus Replication

doi: 10.1371/journal.pone.0124651

Figure Lengend Snippet: ( A - D ) A549 cells were transiently transfected with specific siRNA targeting FGFR1, FGFR4, or negative control siRNA. Forty-eight hours later, A549 cells were infected with PR8 virus at an MOI of 1. The cell culture supernatants and cell lysates were obtained at 24 hpi. Influenza virus M1 mRNA expression in A549 cells with PR8 ( A ) or H5N1 ( B ) infection was detected using real-time PCR. Progeny virus titers of PR8 ( C ) or H5N1 ( D ) were determined using MDCK cells with the TCID 50 assay. ( E ) The knockdown efficiencies of FGFR1 and FGFR4 by target siRNA were tested using real-time PCR. ( F ) Protein expressions of FGFR1 and FGFR4 were detected using specific antibodies by Western blotting assay. All graphs represent the means ± s.e.m. (n = 3). Values of P<0.01 **and P<0.001 *** were considered statistically highly significant.

Article Snippet: Membranes with total FGFR proteins were blocked by 5% (w/v) non-fat dry milk (GE Healthcare), while those with phosphorylated FGFR1 protein were treated with 5% (w/v) bovine serum albumin (BSA) for 1 h at room temperature and then incubated overnight at 4°C with primary antibodies including anti-FGFR1 polyclonal antibody (1:500 dilution, Cell Signaling Technology), anti-phosphorylated FGFR1 polyclonal antibody (1:1000 dilution, Lianke Biotechnology Ltd.), anti-FGFR4 monoclonal antibody (1:1000 dilution, Abcam; Epitomics), and anti-β-actin monoclonal antibody (1:5000 dilution, Sigma-Aldrich).

Techniques: Transfection, Negative Control, Infection, Virus, Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Knockdown, Western Blot

Journal: PLoS ONE

Article Title: A Functional Role of Fibroblast Growth Factor Receptor 1 (FGFR1) in the Suppression of Influenza A Virus Replication

doi: 10.1371/journal.pone.0124651

Figure Lengend Snippet: ( A - C ) A549 cells were transfected with FGFR1 siRNA#1, FGFR4 siRNA#1, and negative control siRNA. After 48 h of transduction, A549 cells were incubated with PR8 virus at an MOI of 0.01 for 4 h, followed by indirect immunofluorescence assays. A549 cells were stained with anti-influenza A virus NP antibodies (green) and Hoechst 33342 (nucleus, blue). ( D ) The data of PR8-infected cells were presented as the percentages of NP-positive cells to the total number of cells. The bars represent the means ± s.e.m. (n = 3). P<0.001 *** were considered statistically highly significant.

Article Snippet: Membranes with total FGFR proteins were blocked by 5% (w/v) non-fat dry milk (GE Healthcare), while those with phosphorylated FGFR1 protein were treated with 5% (w/v) bovine serum albumin (BSA) for 1 h at room temperature and then incubated overnight at 4°C with primary antibodies including anti-FGFR1 polyclonal antibody (1:500 dilution, Cell Signaling Technology), anti-phosphorylated FGFR1 polyclonal antibody (1:1000 dilution, Lianke Biotechnology Ltd.), anti-FGFR4 monoclonal antibody (1:1000 dilution, Abcam; Epitomics), and anti-β-actin monoclonal antibody (1:5000 dilution, Sigma-Aldrich).

Techniques: Transfection, Negative Control, Transduction, Incubation, Virus, Immunofluorescence, Staining, Infection

Journal: PLoS ONE

Article Title: A Functional Role of Fibroblast Growth Factor Receptor 1 (FGFR1) in the Suppression of Influenza A Virus Replication

doi: 10.1371/journal.pone.0124651

Figure Lengend Snippet: A549 cells were infected with recombinant lentivirus expressing FGFR1, FGFR4, or GFP (as a control). After 48 h, A549 cells were infected with PR8 virus (MOI = 1). The supernatants and lysates of A549 cells were harvested after 24 h virus infection. ( A , B ) Influenza M1 mRNA levels in A549 cells with PR8 and H5N1 were detected using real-time PCR. ( C , D ) Progeny virus titers of PR8 and H5N1 were determined as described previously. ( E , F ) FGFR1 and FGFR4 expression efficiencies were detected using real-time PCR and Western blotting. All graphs present the means ± s.e.m. (n = 3). Values of P<0.05 * were considered statistically significant and P<0.001 *** statistically highly significant.

Article Snippet: Membranes with total FGFR proteins were blocked by 5% (w/v) non-fat dry milk (GE Healthcare), while those with phosphorylated FGFR1 protein were treated with 5% (w/v) bovine serum albumin (BSA) for 1 h at room temperature and then incubated overnight at 4°C with primary antibodies including anti-FGFR1 polyclonal antibody (1:500 dilution, Cell Signaling Technology), anti-phosphorylated FGFR1 polyclonal antibody (1:1000 dilution, Lianke Biotechnology Ltd.), anti-FGFR4 monoclonal antibody (1:1000 dilution, Abcam; Epitomics), and anti-β-actin monoclonal antibody (1:5000 dilution, Sigma-Aldrich).

Techniques: Infection, Recombinant, Expressing, Control, Virus, Real-time Polymerase Chain Reaction, Western Blot

Journal: PLoS ONE

Article Title: A Functional Role of Fibroblast Growth Factor Receptor 1 (FGFR1) in the Suppression of Influenza A Virus Replication

doi: 10.1371/journal.pone.0124651

Figure Lengend Snippet: (A) The lysates of A549 cells with PR8 virus infection were harvested for the indicated times. FGFR1 phosphorylation levels were determined by Western blotting with phospho-specific antibodies. Densitometric analysis of the phosphorylated/total FGFR1 ratio was shown as fold change. (B-D) A549 cells were pretreated with PD173074 (FGFR1 kinase inhibitor) at indicated concentrations for 30 min, and then infected with PR8 (indicated-dose inhibitor was also added to virus diluent). After 1 h, the medium was replaced with fresh medium supplemented with PD173074. (B) The cell lysates were collected at 12 hpi and then subjected to Western blotting for FGFR1 phosphorylation detection. The phosphorylated/total FGFR1 ratio indicated the fold change of FGFR1 phosphorylation. (C) Influenza M1 mRNA in PR8 infected-A549 cells with PD173074 treatment as indicated was detected by real-time PCR. (D) MTT assay of A549 cells with PD173074 treatment as indicated with or without PR8 infection. The results were expressed as means ± s.e.m. (n = 3). Values of P<0.05 * were considered statistically significant and P<0.001 *** considered statistically highly significant.

Article Snippet: Membranes with total FGFR proteins were blocked by 5% (w/v) non-fat dry milk (GE Healthcare), while those with phosphorylated FGFR1 protein were treated with 5% (w/v) bovine serum albumin (BSA) for 1 h at room temperature and then incubated overnight at 4°C with primary antibodies including anti-FGFR1 polyclonal antibody (1:500 dilution, Cell Signaling Technology), anti-phosphorylated FGFR1 polyclonal antibody (1:1000 dilution, Lianke Biotechnology Ltd.), anti-FGFR4 monoclonal antibody (1:1000 dilution, Abcam; Epitomics), and anti-β-actin monoclonal antibody (1:5000 dilution, Sigma-Aldrich).

Techniques: Virus, Infection, Phospho-proteomics, Western Blot, Real-time Polymerase Chain Reaction, MTT Assay

Journal: PLoS ONE

Article Title: A Functional Role of Fibroblast Growth Factor Receptor 1 (FGFR1) in the Suppression of Influenza A Virus Replication

doi: 10.1371/journal.pone.0124651

Figure Lengend Snippet: A549 cells with FGFR1, FGFR4, or GFP overexpression were pretreated with or without sialidase, and then infected with influenza A/PR8 ( A ) or H5N1 ( B ). The procedures were described in detail in the Methods, virus binding and internalization assay. Influenza virus NP was detected by Western blotting using anti-influenza NP antibodies. β-actin protein was used as an internal control. The ratio of NP/β-actin was determined based on densitometric analysis. The results were expressed as means ± s.e.m. (n = 3). Values of P<0.05 * were considered statistically significant and P<0.01 ** highly significant.

Article Snippet: Membranes with total FGFR proteins were blocked by 5% (w/v) non-fat dry milk (GE Healthcare), while those with phosphorylated FGFR1 protein were treated with 5% (w/v) bovine serum albumin (BSA) for 1 h at room temperature and then incubated overnight at 4°C with primary antibodies including anti-FGFR1 polyclonal antibody (1:500 dilution, Cell Signaling Technology), anti-phosphorylated FGFR1 polyclonal antibody (1:1000 dilution, Lianke Biotechnology Ltd.), anti-FGFR4 monoclonal antibody (1:1000 dilution, Abcam; Epitomics), and anti-β-actin monoclonal antibody (1:5000 dilution, Sigma-Aldrich).

Techniques: Over Expression, Infection, Virus, Binding Assay, Western Blot, Control

Journal: Osteoarthritis and cartilage / OARS, Osteoarthritis Research Society

Article Title: SEQUENTIAL EXPOSURE TO FIBROBLAST GROWTH FACTORS (FGF) 2, 9 AND 18 ENHANCES hMSC CHONDROGENIC DIFFERENTIATION

doi: 10.1016/j.joca.2014.11.013

Figure Lengend Snippet: (A) Sox9 mRNA (n=4) at the end of 14 days of expansion (Exp. - d0) and at early chondrogenic differentiation in aggregate cultures (Diff. – d3) with and without FGF2. Statistical comparisons made against control-expanded data at d0 and d3 independently (left panel) and against d0 for both groups (right panel). (B) Sox9 protein at the end of 14 days of expansion in control and FGF2-rich medium. (C) FGFR1 and FGFR3 (n=3) gene expression profiles (assessed by qRT-PCR) evaluated after 14 days of expansion (d0) and subsequent aggregate culture for 21 days. (#): Statistical significance (p<0.0001) when data compared to control-expanded at d0; (&): Statistical significance (p<0.0001) when data compared to FGF2-expanded at d0. Statistical significances (p values) when Control and FGF2-expanded cells are compared at a particular time-point are shown in the graph for clarity. Gene expression analysis was performed for all four donors (Sox9) and three donors (FGFRs) using triplicate samples.

Article Snippet: Chondrogenic induction Control and FGF2-expanded cells were cultured in aggregates 4 , 5 with complete chondrogenic medium (DMEM-HG supplemented with 1% ITS, 10 −7 M dexamethasone, 1 mM sodium pyruvate, 120 μM ascorbic acid-2 phosphate, 100 μM non-essential amino acids and 10 ng/ml TGF-β1), in the presence of 10 ng/ml rhFGF18, 10 ng/ml rhFGF9, 10 ng/ml mutant ligands that exclusively signal through FGFR3 (FGF9v1 and FGF18v3) and neutralizing antibodies against FGFR1 (0.1 μg/ml) and FGFR3 (0.5 μg/ml) (Procore; Ness Ziona, Israel) starting at different times of the chondroinductive program.

Techniques: Control, Gene Expression, Quantitative RT-PCR

Journal: Osteoarthritis and cartilage / OARS, Osteoarthritis Research Society

Article Title: SEQUENTIAL EXPOSURE TO FIBROBLAST GROWTH FACTORS (FGF) 2, 9 AND 18 ENHANCES hMSC CHONDROGENIC DIFFERENTIATION

doi: 10.1016/j.joca.2014.11.013

Figure Lengend Snippet: Control and FGF2-expanded cells (n=3) were subjected to FGF9 or FGF18 stimulation from d0 to d21 of differentiation induction in pellet cultures, where d0 data corresponds to expanded cells just before aggregate formation. Pellet cultures were harvested at day 5, 10, 15 and 21, and qRT-PCR performed to evaluate FGFR3 and FGFR1 gene expression (A & B) and FGFR3:FGFR1 ratio (C). Results with non-stimulated Control (black dots) and FGF2-expanded cells (white dots) are shown again as in Fig 1C for reference (statistical differences presented in Fig 1C). Data obtained at every time point with FGF9 and FGF18 stimulation were compared with Control-expanded and FGF2-expanded cells at the same point, and the statistical differences (p values) shown in the graph for clarity. All other comparison were p>0.1.

Article Snippet: Chondrogenic induction Control and FGF2-expanded cells were cultured in aggregates 4 , 5 with complete chondrogenic medium (DMEM-HG supplemented with 1% ITS, 10 −7 M dexamethasone, 1 mM sodium pyruvate, 120 μM ascorbic acid-2 phosphate, 100 μM non-essential amino acids and 10 ng/ml TGF-β1), in the presence of 10 ng/ml rhFGF18, 10 ng/ml rhFGF9, 10 ng/ml mutant ligands that exclusively signal through FGFR3 (FGF9v1 and FGF18v3) and neutralizing antibodies against FGFR1 (0.1 μg/ml) and FGFR3 (0.5 μg/ml) (Procore; Ness Ziona, Israel) starting at different times of the chondroinductive program.

Techniques: Control, Quantitative RT-PCR, Gene Expression, Comparison

Journal: Osteoarthritis and cartilage / OARS, Osteoarthritis Research Society

Article Title: SEQUENTIAL EXPOSURE TO FIBROBLAST GROWTH FACTORS (FGF) 2, 9 AND 18 ENHANCES hMSC CHONDROGENIC DIFFERENTIATION

doi: 10.1016/j.joca.2014.11.013

Figure Lengend Snippet: FGF2-expanded cells were stimulated with ligands or mutant variants for 7 days starting at d14 (until d21). Receptor neutralizing antibodies were added 48 hours in advance (d12) to assure blocking before ligands were administered. Pellet shown in the insert (control) corresponds to black dots shown on B). (A) Histological assessment of representative pellets confirming the results with FGF ligands and mutants (as presented in Fig. 3 – d14), and the determinant role of FGFR3 in this effect, (B) GAG/DNA quantification and gene expression analysis of type 2 collagen and aggrecan, assessed by qRT-PCR performed at day 21. Statistical significance (#) was obtained comparing all values to control pellet (black dots) with the following p values: for GAG/DNA: β+9 (0.0007), β+9v1 (<0.0001), β+9+R1 (0.0004), β+9+R3 (0.0012), β+18 (0.0008), β+18v3 (0.0002), β+18+R1 (0.0091), β+18+R3 (0.0100). For Aggrecan expression: β+9v1 (<0.0013) and β+18v3 (0.0003). For Col2 expression β+9v1 (0.0002), β+18v3 (0.0001), β+18+R1 (0.0009). All other comparisons were p > 0.1. The effects of blocking FGFR1 and FGFR3 receptors when compared with respective growth factor alone are shown in the graph with significant differences (p values) displayed in color. N=4.

Article Snippet: Chondrogenic induction Control and FGF2-expanded cells were cultured in aggregates 4 , 5 with complete chondrogenic medium (DMEM-HG supplemented with 1% ITS, 10 −7 M dexamethasone, 1 mM sodium pyruvate, 120 μM ascorbic acid-2 phosphate, 100 μM non-essential amino acids and 10 ng/ml TGF-β1), in the presence of 10 ng/ml rhFGF18, 10 ng/ml rhFGF9, 10 ng/ml mutant ligands that exclusively signal through FGFR3 (FGF9v1 and FGF18v3) and neutralizing antibodies against FGFR1 (0.1 μg/ml) and FGFR3 (0.5 μg/ml) (Procore; Ness Ziona, Israel) starting at different times of the chondroinductive program.

Techniques: Mutagenesis, Blocking Assay, Control, Gene Expression, Quantitative RT-PCR, Expressing

Journal: Osteoarthritis and cartilage / OARS, Osteoarthritis Research Society

Article Title: SEQUENTIAL EXPOSURE TO FIBROBLAST GROWTH FACTORS (FGF) 2, 9 AND 18 ENHANCES hMSC CHONDROGENIC DIFFERENTIATION

doi: 10.1016/j.joca.2014.11.013

Figure Lengend Snippet: (A) Gene expression profile of hypertrophy markers showing an early onset of terminal differentiation of FGF2-expanded cells compared with control-expanded cells. P values are shown for each gene in the graph for clarity. (B) Gene expression profile assessment of FGF2-expanded cells (n=4) stimulated with FGF9, FGF18 and variants along with blocking FGFR3 (+R3) or FGFR1 (+R1) antibodies for two weeks (d14 to d28). Compared to control pellets without stimulation (β), statistical significance (p<0.0001) was obtained for the majority of conditions (#) in all four genes. The effects of blocking FGFR1 and FGFR3 receptors when compared with respective growth factor alone are shown in the graph with significant differences (p values) displayed in color. N=4.

Article Snippet: Chondrogenic induction Control and FGF2-expanded cells were cultured in aggregates 4 , 5 with complete chondrogenic medium (DMEM-HG supplemented with 1% ITS, 10 −7 M dexamethasone, 1 mM sodium pyruvate, 120 μM ascorbic acid-2 phosphate, 100 μM non-essential amino acids and 10 ng/ml TGF-β1), in the presence of 10 ng/ml rhFGF18, 10 ng/ml rhFGF9, 10 ng/ml mutant ligands that exclusively signal through FGFR3 (FGF9v1 and FGF18v3) and neutralizing antibodies against FGFR1 (0.1 μg/ml) and FGFR3 (0.5 μg/ml) (Procore; Ness Ziona, Israel) starting at different times of the chondroinductive program.

Techniques: Gene Expression, Control, Blocking Assay

Journal: Osteoarthritis and cartilage / OARS, Osteoarthritis Research Society

Article Title: SEQUENTIAL EXPOSURE TO FIBROBLAST GROWTH FACTORS (FGF) 2, 9 AND 18 ENHANCES hMSC CHONDROGENIC DIFFERENTIATION

doi: 10.1016/j.joca.2014.11.013

Figure Lengend Snippet: Induced terminal differentiation of FGF2-expanded hMSC evaluated cytomorphologically6,7 (top row) and by the activity of the hypertrophy-specific marker Alkaline Phosphatase (ALP) in sections (lower row) and quantified (dA/min = changes of Absorbance at A405 in time within the linear range) in the medium6 (insert). Compared to control, all conditions with hypertrophy induction are statistically different (p<0.0001). Compared to hypertrophy alone (H): the addition of FGF9 (p=0.0441) and FGF18 (p=0.0280) delayed the appearance of those enhanced terminal differentiation characteristics. Blocking FGFR3 prevented this effect (with FGF9: p=0.1975, and with FGF18: p=0.4863), while blocking FGFR1 did not (with FGF9: p=0.0459, and with FGF18: p=0.0478). This effect of blocking the receptors is further supported when compared to the growth factors alone: p values shown in graph for clarity and significant differences displayed in color. N=4.

Article Snippet: Chondrogenic induction Control and FGF2-expanded cells were cultured in aggregates 4 , 5 with complete chondrogenic medium (DMEM-HG supplemented with 1% ITS, 10 −7 M dexamethasone, 1 mM sodium pyruvate, 120 μM ascorbic acid-2 phosphate, 100 μM non-essential amino acids and 10 ng/ml TGF-β1), in the presence of 10 ng/ml rhFGF18, 10 ng/ml rhFGF9, 10 ng/ml mutant ligands that exclusively signal through FGFR3 (FGF9v1 and FGF18v3) and neutralizing antibodies against FGFR1 (0.1 μg/ml) and FGFR3 (0.5 μg/ml) (Procore; Ness Ziona, Israel) starting at different times of the chondroinductive program.

Techniques: Activity Assay, Marker, Control, Blocking Assay

Journal: Osteoarthritis and cartilage / OARS, Osteoarthritis Research Society

Article Title: SEQUENTIAL EXPOSURE TO FIBROBLAST GROWTH FACTORS (FGF) 2, 9 AND 18 ENHANCES hMSC CHONDROGENIC DIFFERENTIATION

doi: 10.1016/j.joca.2014.11.013

Figure Lengend Snippet: FGF2 is used to increase cell number and prime the cells for chondrogenesis (via Sox9 early appearance) during expansion, followed by stimulation with TGF-β to start chondrogenesis and either FGF9, FGF18 or FGFR3-specific ligands at day 14 (high FGFR3:FGFR1 ratio) to induce ECM secretion and delay the appearance of a hypertrophic phenotype. The program is intended to induce hMSC differentiation into a more stable pre-hypertrophic phenotype, typical of articular cartilage chondrocytes. Figure made with images available at Servier Medical Art (www.servier.fr).

Article Snippet: Chondrogenic induction Control and FGF2-expanded cells were cultured in aggregates 4 , 5 with complete chondrogenic medium (DMEM-HG supplemented with 1% ITS, 10 −7 M dexamethasone, 1 mM sodium pyruvate, 120 μM ascorbic acid-2 phosphate, 100 μM non-essential amino acids and 10 ng/ml TGF-β1), in the presence of 10 ng/ml rhFGF18, 10 ng/ml rhFGF9, 10 ng/ml mutant ligands that exclusively signal through FGFR3 (FGF9v1 and FGF18v3) and neutralizing antibodies against FGFR1 (0.1 μg/ml) and FGFR3 (0.5 μg/ml) (Procore; Ness Ziona, Israel) starting at different times of the chondroinductive program.

Techniques: