Journal: bioRxiv

Article Title: The Ets transcription factor ETV4 regulates FGF1-dependent proliferation and glycolysis in ER-positive breast cancer

doi: 10.64898/2026.01.13.699240

Figure Lengend Snippet: (A) ETV4 expression in breast tumors and paired normal breast tissue from people with breast cancer. Data obtained from tnmplot.com. Mann-Whitney test determined significance. (B) High ETV4 expression (RNA seq) associates with lower overall survival for people with breast cancer. HR=1.44 (1.14-1.82). (C) Tumors with high ETV4 expression (mRNA z-score >1.5) are more likely to be ER-negative than ER-positive. Chi-squared test q-value <0.0001. (D) High ETV4 expression (RNA seq) associates with lower overall survival for patients with ER-positive breast cancer. HR=1.39 (1.05-1.85). (E) High ETV4 expression (array) associates with lower recurrence-free survival for patients with lymph node-positive ER-positive breast cancer. HR=1.3 (1.031-1.628). (F) ETV4 expression in ER-positive breast tumors stratified by pathologic complete response after aromatase inhibitors using data from ROCplotter.com. (G) ETV4 expression in ER-positive tumors stratified by pathologic complete response to any chemotherapy using data from ROCplotter.com. Mann-Whitney test determined significance for f and g . (H) ETV4 expression (RNA seq) in UCD12 PDX tumors from lean or obese female mice. (I) ETV4 expression (array) in tumors from patients with ER-positive breast cancer (data from GSE24185). (J) Pearson correlation between ETV4 and FGF1 expression in tumors from patients classified as obese, overweight, or lean based on BMI (data from GSE24185). (K) Expression of ETV4 in ER-positive breast cancer cells with or without FGF1 treatment. MCF7 Parental (M7P) or TAMR (M7T); UCD12 (U12). (L) Representative western blot showing ETV4 expression in ER-positive breast cancer cells with or without FGF1 treatment.

Article Snippet: Recombinant human FGF1 was purchased from R&D Systems and was diluted in 0.01% bovine serum albumin (BSA) in PBS and used at a final concentration of 5 ng/mL.

Techniques: Expressing, MANN-WHITNEY, RNA Sequencing, Western Blot

Journal: bioRxiv

Article Title: The Ets transcription factor ETV4 regulates FGF1-dependent proliferation and glycolysis in ER-positive breast cancer

doi: 10.64898/2026.01.13.699240

Figure Lengend Snippet: (A) Q-PCR ( left ) and immunoblot ( right ) analysis of ETV4 in MCF7-P cells. (B) Q-PCR ( left ) and immunoblot ( right ) analysis of ETV4 in MCF7-TAMR cells. (C) Representative images of the final timepoint (left) and growth rates (right) of MCF7-P control or ETV4-knockdown cells treated with vehicle or FGF1. (D) Area confluence relative to control vehicle time 0 of cells at the final timepoint following treatment of MCF7-P cells. Two-way ANOVA testing for main effects of ETV4 knockdown or FGF1 treatment or interaction was performed. P-values indicate post-hoc multiple testing for specific differences between pre-defined comparisons. (E) Representative images of the final timepoint (left) and growth rates (right) of MCF7-TAMR control or ETV4-knockdown cells treated with vehicle or FGF1. (F) Area confluence relative to control vehicle time 0 of cells at the final timepoint following treatment of MCF7-TAMR cells. Two-way ANOVA testing for main effects of ETV4 knockdown or FGF1 treatment or interaction was performed. P-values indicate post-hoc multiple testing for specific differences between pre-defined comparisons. (G) Dose-response curve and interpolated IC 50 values of tamoxifen treatment in MCF7-P control and ETV4 knockdown cells following treatment with or without FGF1. (H) Dose-response curve and interpolated IC 50 values of tamoxifen treatment in MCF7-TAMR control and ETV4 knockdown cells following treatment with or without FGF1.

Article Snippet: Recombinant human FGF1 was purchased from R&D Systems and was diluted in 0.01% bovine serum albumin (BSA) in PBS and used at a final concentration of 5 ng/mL.

Techniques: Western Blot, Control, Knockdown

Journal: bioRxiv

Article Title: The Ets transcription factor ETV4 regulates FGF1-dependent proliferation and glycolysis in ER-positive breast cancer

doi: 10.64898/2026.01.13.699240

Figure Lengend Snippet: (A) Expression of ETV4 gene ( left ) and protein ( right ) following knockdown in MCF7-P cells. (B) Expression of ETV4 gene ( left ) and protein ( right ) following knockdown in MCF7-TAMR cells. (C) Representative images of the final timepoint (left) and growth rates (right) of MCF7-P control or ETV4-overexpressing cells treated with vehicle or FGF1. (D) Area confluence relative to control vehicle time 0 of cells at the final timepoint following treatment of MCF7-P cells. Two-way ANOVA testing for main effects of ETV4 overexpression or FGF1 treatment or interaction was performed. P-values indicate post-hoc multiple testing for specific differences between pre-defined comparisons. (E) Representative images of the final timepoint (left) and growth rates (right) of MCF7-TAMR control or ETV4-overexpressing cells treated with vehicle or FGF1. (F) Area confluence relative to control vehicle time 0 of cells at the final timepoint following treatment of MCF7-TAMR cells. Two-way ANOVA testing for main effects of ETV4 overexpression or FGF1 treatment or interaction was performed. P-values indicate post-hoc multiple testing for specific differences between pre-defined comparisons. (G) Dose-response curve and interpolated IC 50 values of tamoxifen treatment in MCF7-P control and ETV4 overexpressing cells following treatment with or without FGF1. (H) Dose-response curve and interpolated IC 50 values of tamoxifen treatment in MCF7-TAMR control and ETV4 overexpressing cells following treatment with or without FGF1. (I) Dose-response curve and interpolated IC 50 values of BGJ398 treatment in MCF7-P and MCF7-TAMR control and ETV4 overexpressing cells.

Article Snippet: Recombinant human FGF1 was purchased from R&D Systems and was diluted in 0.01% bovine serum albumin (BSA) in PBS and used at a final concentration of 5 ng/mL.

Techniques: Expressing, Knockdown, Control, Over Expression

Journal: bioRxiv

Article Title: The Ets transcription factor ETV4 regulates FGF1-dependent proliferation and glycolysis in ER-positive breast cancer

doi: 10.64898/2026.01.13.699240

Figure Lengend Snippet: (A) Bubble plot of gene set enrichment analysis (GSEA) showing enriched pathways in MCF7-TAMR control, ETV4 knockdown, and ETV4-overexpressing cells with or without FGF1 stimulation. (B) Hallmarks of Cancer enrichment plot illustrating the pathways represented by genes that are downregulated by ≥2-fold (adjusted p-value) in ETV4 knockdown compared with control vehicle-treated MCF7-TAMR cells. Bar height reflects −log10 adjusted p-value, with dashed circles indicating significance thresholds. (C) Volcano plot comparing ETV4 knockdown versus control vehicle-treated MCF7-TAMR cells, highlighting downregulated genes involved in reprogramming energy metabolism. Blue points denote significantly downregulated genes. Blue points denote significantly downregulated genes that correspond to the significant cancer hallmarks. (D) Hallmarks of Cancer enrichment plot illustrating the pathways represented by genes that are upregulated by ≥1.58-fold (adjusted p-value) in MCF7-TAMR ETV4 overexpressing cells treated with FGF1 vs vehicle controls. Bar height reflects −log10 adjusted p-value, with dashed circles indicating significance thresholds, including sustaining proliferative signaling. (E) Volcano plot comparing genes significantly altered in MCF7-TAMR ETV4 overexpressing cells treated with or without FGF1. Red points denote significantly upregulated genes that correspond to the significant cancer hallmarks.

Article Snippet: Recombinant human FGF1 was purchased from R&D Systems and was diluted in 0.01% bovine serum albumin (BSA) in PBS and used at a final concentration of 5 ng/mL.

Techniques: Control, Knockdown

Journal: bioRxiv

Article Title: The Ets transcription factor ETV4 regulates FGF1-dependent proliferation and glycolysis in ER-positive breast cancer

doi: 10.64898/2026.01.13.699240

Figure Lengend Snippet: (A-B) Heatmaps showing mRNA expression levels of glycolytic pathway genes (HK2, PFKP, PGK1, ENO1, and LDHA) under vehicle and FGF1-treated conditions in MCF7-P ( a ) and MCF7-TAMR ( b ) control and ETV4 knockdown cells, respectively. Data are expressed as fold change versus the average of vehicle treated cells for each gene, showing 3 replicates per group. (C) Seahorse metabolic flux analysis showing the kinetic graph of oxygen consumption rate (OCR) in MCF7-P cells. (D-G) Metabolic parameters including basal respiration ( d ), maximal respiration ( e ), ATP-production coupled respiration ( f ), and ECAR ( g ) in control and ETV4 knockdown MCF7-P cells, upon vehicle and FGF1 stimulation. Data analyzed using a 2-way ANOVA testing for main effects of ETV4 or FGF1 treatment or interactions. P-values denote post-hoc analysis of specific comparisons. (H) Seahorse metabolic flux analysis showing the kinetic graph of oxygen consumption rate (OCR) in MCF7-TAMR cells. (I-L) Metabolic parameters including basal respiration ( i ), maximal respiration ( j ), ATP-production coupled respiration ( k ), and ECAR ( l ) in control and ETV4 knockdown MCF7-TAMR cells, upon vehicle and FGF1 stimulation. Data were analyzed using a 2-way ANOVA testing for main effects of ETV4 or FGF1 treatment or interactions. P-values denote post-hoc analysis of specific comparisons. All Seahorse data were normalized to total protein in each well. N=16-24 replicates per measure.

Article Snippet: Recombinant human FGF1 was purchased from R&D Systems and was diluted in 0.01% bovine serum albumin (BSA) in PBS and used at a final concentration of 5 ng/mL.

Techniques: Expressing, Control, Knockdown

Journal: bioRxiv

Article Title: The Ets transcription factor ETV4 regulates FGF1-dependent proliferation and glycolysis in ER-positive breast cancer

doi: 10.64898/2026.01.13.699240

Figure Lengend Snippet: (A-B) Heatmaps showing mRNA expression levels of glycolytic pathway genes (HK2, PFKP, PGK1, ENO1, and LDHA) under vehicle and FGF1-treated conditions in MCF7-P ( a ) and MCF7-TAMR ( b ) control and ETV4 overexpressing cells, respectively. Data are expressed as fold change versus the average of vehicle treated cells for each gene, showing 3 replicates per group. (C) Seahorse metabolic flux analysis showing the kinetic graph of oxygen consumption rate (OCR) in MCF7-P cells. (D-G) Metabolic parameters including basal respiration ( d ), maximal respiration ( e ), ATP-production coupled respiration ( f ), and ECAR ( g ) in control and ETV4 overexpressing MCF7-P cells upon vehicle and FGF1 stimulation. Data were analyzed using a 2-way ANOVA testing for main effects of ETV4 or FGF1 treatment or interactions. P-values denote post-hoc analysis of specific comparisons. (H) Seahorse metabolic flux analysis showing the kinetic graph of oxygen consumption rate (OCR) in MCF7-TAMR cells. (I-L) Metabolic parameters including basal respiration ( i ), maximal respiration ( j ), ATP-production coupled respiration ( k ), and ECAR ( l ) in control and ETV4 knockdown MCF7-TAMR cells, upon vehicle and FGF1 stimulation. Data were analyzed using a 2-way ANOVA testing for main effects of ETV4 or FGF1 treatment or interactions. P-values denote post-hoc analysis of specific comparisons. All Seahorse data were normalized to total protein in each well. N=16-24 replicates per measure.

Article Snippet: Recombinant human FGF1 was purchased from R&D Systems and was diluted in 0.01% bovine serum albumin (BSA) in PBS and used at a final concentration of 5 ng/mL.

Techniques: Expressing, Control, Knockdown

Journal: Molecular Biomedicine

Article Title: Engineered fibroblast growth factor 1 variants uncouple glucose-lowering effects from mitogenic activity with therapeutic potential for type 2 diabetes

doi: 10.1186/s43556-025-00398-w

Figure Lengend Snippet: Effect of introducing point mutations on the biological activity of FGF1. a Serum-starved NIH 3T3 cells were treated with 10 ng/mL FGF1 variants for 15 min in the presence of heparin (10 U/mL). Activation of the downstream cascade was detected by immunoblotting using the following antibodies: anti-phospho-FRS2 (pFRS2) and anti-phospho-ERK1/2 (pERK1/2). Anti-ERK1/2 and anti-vinculin antibodies were used to confirm equal loading. Representative results are shown (n ≥ 3). The vertical lines in the last WB panel show the deleted wells. The original membranes, together with the method of trimming, are presented in Fig. S2. Densitometric analysis of pERK/ERK is presented in Fig. S3. b Effect of 20-h FGF1 variants stimulation (20 ng/mL) in the presence of 10 U/mL heparin on glucose uptake by 3T3-L1 adipocytes. Data are presented as mean ± SEM, n = 4. Statistical significance: * p ≤ 0.05; ** p ≤ 0.01 and *** p ≤ 0.001

Article Snippet: Human FGF1 ELISA Kit was purchased from R&D Systems (#DFA00B).

Techniques: Activity Assay, Activation Assay, Western Blot

Journal: Molecular Biomedicine

Article Title: Engineered fibroblast growth factor 1 variants uncouple glucose-lowering effects from mitogenic activity with therapeutic potential for type 2 diabetes

doi: 10.1186/s43556-025-00398-w

Figure Lengend Snippet: Impaired activation of signaling pathways by FGF1 variants due to reduced affinity for the FGFR1 (IIIc) receptor. a Serum-starved NIH 3T3 cells were stimulated with 10 ng/mL FGF1 variants in the presence of heparin (10 U/mL) for 15 min, and activation of downstream signaling cascades was detected by immunoblotting using the following antibodies: anti-phospho-FGFR (pFGFR), anti-phospho-PLCγ (pPLCγ), anti-phosphoFRS2 (pFRS2), anti-phospho-ERK1/2 (pERK1/2). Anti-ERK1/2, anti-FGFR1, anti-PLCγ and anti-γTubulin antibodies were used to confirm equal loading. Representative results are shown. Densitometric analysis is presented as mean ± SEM, n = 3/4. Statistical significance: * p ≤ 0.05; ** p ≤ 0.01 and *** p ≤ 0.001. b BLI analysis of the affinity of FGF1 variants for FGFR1-Fc (IIIc isoform). FGFR1-Fc was immobilized on a Protein A sensor and its interactions (association and dissociation) with selected FGF1 mutants were analyzed in the concentration range of 100–800 nM. Curves obtained by global fitting are marked in red. Representative results are shown (n ≥ 3). The equilibrium dissociation constant (K D ) was calculated from the saturation binding curve

Article Snippet: Human FGF1 ELISA Kit was purchased from R&D Systems (#DFA00B).

Techniques: Activation Assay, Protein-Protein interactions, Western Blot, Concentration Assay, Binding Assay

Journal: Molecular Biomedicine

Article Title: Engineered fibroblast growth factor 1 variants uncouple glucose-lowering effects from mitogenic activity with therapeutic potential for type 2 diabetes

doi: 10.1186/s43556-025-00398-w

Figure Lengend Snippet: Effect of introduced mutations on the long-term activity of FGF1. a Induction of Glut1 expression after stimulation with FGF1 variants. 3T3-L1 cells were treated with 100 ng/mL FGF1 muteins in the presence of 10 U/mL heparin for 24 h, followed by WB analysis with anti-Glut1 and anti-γTubulin antibodies, data are presented as mean ± SEM, n = 6. Statistical significance: * p ≤ 0.05; ** p ≤ 0.01 and *** p ≤ 0.001. b Normalized thermal denaturation curves of FGF1 variants monitored by ellipticity changes (λ = 227 nm). c Degradation of FGF1 variants in the presence of adipocytes. Serum-starved 3T3-L1 adipocytes were incubated with 1 µg/mL FGF1 variants. The progress of proteolysis on subsequent days was monitored by immunoblotting with anti-FGF1 antibody. Representative results are shown ( n = 5). Densitometric analysis of proteolysis of FGF1 variants is presented as the ratio the intensity of the upper band to the intensity of the whole amount of protein. Mean ± SEM are shown, n = 5. Statistical significance: * p ≤ 0.05; ** p ≤ 0.01 and *** p ≤ 0.001

Article Snippet: Human FGF1 ELISA Kit was purchased from R&D Systems (#DFA00B).

Techniques: Activity Assay, Expressing, Incubation, Western Blot

Journal: Molecular Biomedicine

Article Title: Engineered fibroblast growth factor 1 variants uncouple glucose-lowering effects from mitogenic activity with therapeutic potential for type 2 diabetes

doi: 10.1186/s43556-025-00398-w

Figure Lengend Snippet: Mitogenic activity of selected FGF1 variants in different cell types. Mitogenic activity of selected FGF1 variants was assessed by stimulating serum-starved C2C12, MCF7 or 4MBr-5 cells with FGF1 mutants in the concentration range 0.1–100 ng/ml in the presence of 10 U/ml heparin for 48 h, 72 h or 96 h, respectively. Cell viability was assessed by PrestoBlue cell viability assay a or by CellTiter-Fluor cell viability assay b . Cell number was determined by counting NucBlue-stained nuclei using an Opera Phenix Plus High-Content Screening System c . Data are presented as mean ± SEM, n = 3. Multiple t-test; statistical significance: * p ≤ 0.05; ** p ≤ 0.01 and *** p ≤ 0.001

Article Snippet: Human FGF1 ELISA Kit was purchased from R&D Systems (#DFA00B).

Techniques: Activity Assay, Concentration Assay, Viability Assay, Staining, High Content Screening

Journal: Molecular Biomedicine

Article Title: Engineered fibroblast growth factor 1 variants uncouple glucose-lowering effects from mitogenic activity with therapeutic potential for type 2 diabetes

doi: 10.1186/s43556-025-00398-w

Figure Lengend Snippet: Metabolic activity of FGF1 variants in vivo. a , b Change in blood glucose levels in db/db mice after a single administration of FGF1 variants (measurements at 0, 6, 18, 24, 30, 48, 72, 96 and 168 h after protein administration). FGF1 variants were administered at a dose of 1 mg/kg body weight. Data were normalized to glucose levels before protein administration and presented as mean ± SEM, n = 7/6. Statistical significance * p ≤ 0.05; ** p ≤ 0.01 and *** p ≤ 0.001; (*) indicates comparison with vehicle; (#) indicates comparison with wild-type protein. c Analysis of mouse body weight 96 h after administration. d Pharmacokinetics of FGF1 variants after a single subcutaneous administration in Wistar Han rats. FGF1 variants were administered subcutaneously at a dose 0.5 mg/kg, and blood samples were collected before injection and at 5 min, 15 min, 30 min and 1, 2, 4, 7, 12, 24, 48 h after injection and protein levels were analyzed by ELISA. Data are presented as mean ± SEM, n = 5

Article Snippet: Human FGF1 ELISA Kit was purchased from R&D Systems (#DFA00B).

Techniques: Activity Assay, In Vivo, Comparison, Drug discovery, Injection, Enzyme-linked Immunosorbent Assay

Journal: Angiogenesis

Article Title: Angiopoietin-2 binds to FGFR2, inhibits FGF-FGFR2 signaling, and delays cutaneous wound healing by inhibiting wound angiogenesis

doi: 10.1007/s10456-025-09988-2

Figure Lengend Snippet: Recombinant Ang2 binds to recombinant FGFR2-Fc. A Lysates prepared from HUVEC in starvation medium for 1 hour and then incubated with FGF1 (5 nM, 5 min) or/and Ang2 (5 nM, 5 min) in the same medium were analyzed by Western blotting; antibodies to p-FGFR Y653/654 and FGFR2 recognize a band at ~130 kDa. Representative of 3 experiments. B Schematic of the pull-down experiment. FGFR2: FGFR2ß (IIIb)-Fc. C Ang2 (0.1 μM) specifically binds to recombinant FGFR2ß (IIIb)-Fc (0.1-0.5 μM) but not to human IgG-Fc (Fc, 0.5 μM). The precipitated proteins were immunoblotted with antibodies to Ang2 (left) or to FGFR2ß (IIIb) (right). The asterisks point to bands specifically identifying Ang2 (left) and FGFR2 (right). Representative of 3 experiments. D Schematic of the pull-down experiment. FGFR2: FGFR2α (IIIc)-Fc. E Ang2 (0.1 μM) does not compete with the binding of FGF1 (0.1-0.4 μM) to FGFR2α (IIIc)-Fc (0.2 μM). IgG-Fc used at 0.2 mM. The precipitates were immunoblotted with antibodies to Ang2, FGF1, or anti-human Fc (hFc). The asterisks point to Ang2 (left), FGF1 (middle), and to FGFR2-Fc or Fc (right). Representative of 3 experiments. F Structural modeling of the Ang2, FGF1 and FGFR2α (IIIb) trimeric complex from amino acid sequences by AlphaFold2-Multimer. Red: predicted structure of Ang2 in the predicted trimeric complex. Pink: Ang2 structure from the crystal structure of Ang2 alone (PDB 1z3s). Blue: predicted structure of FGFR2α (IIIb). Light blue: FGFR2ß (IIIb) structure from the crystal structure of FGFR2ß (IIIb)-FGF1 dimeric complex (PDB 1djs). Yellow: predicted structure of FGF1. Green: FGF1 structure from the crystal structure of FGFR2ß (IIIb)-FGF1 complex (PDB 1djs). The three predicted protein structures are largely consistent when superimposed on the respective crystal structures. The predicted structure suggests that Ang2 and FGF1 interact with FGFR2 at distinct sites. G Structural models of the Ang2, FGF1 and FGFR2α (IIIc) trimeric complex (left) and the Ang2, FGF2 and FGFR2α (IIIc) trimeric complex (right) from amino acid sequences by AlphaFold3

Article Snippet: Cells were then incubated with 100–1000 ng/ml recombinant Ang2 (expressed with a C-terminal His-tag in CHO cells; Biolegend, No. 753106) or/and 3 or 5 nM FGF1 (R&D Systems, No. 232-FA) for 5 min, and/or 314.8 nM (10 μg/ml) AMG386 (obtained under material CRADA from Amgen).

Techniques: Recombinant, Incubation, Western Blot, Binding Assay

Journal: Angiogenesis

Article Title: Angiopoietin-2 binds to FGFR2, inhibits FGF-FGFR2 signaling, and delays cutaneous wound healing by inhibiting wound angiogenesis

doi: 10.1007/s10456-025-09988-2

Figure Lengend Snippet: The Ang2 inhibitor AMG386 reduces Ang2 binding to FGFR2-Fc. A Schematic of the pull-down experiment. FGFR2α (IIIc)-Fc. B AMG386 (AMG, 0.2 or 0.05 μM) inhibits the binding of Ang2 (0.1 μM) to FGFR2-Fc (0.2 μM). IgG-Fc (Fc, 0.2 μM). The precipitates were immunoblotted with antibodies to Fc and Ang2. The black asterisk points to FGFR2-Fc (left) and Ang2 (right); the red asterisk points to AMG386. Representative experiment (of three performed). C Overall complex structure of AMG386 (yellow) bound to Ang2 (blue), FGF1 (green), and FGFR2α (IIIb) (pink) is shown as a surface representation. AMG386 occupies the main binding cavity of the complex, highlighting its central role in coordinating interactions with the other proteins. D and E Trp 280 from AMG386 (yellow) forms hydrophobic interactions with nearby residues, including Tyr 135 and His 108 from FGFR2 (pink) and Phe 190 from Ang2 (blue). These interactions include π-stacking between Trp 280 (AMG386) and Phe 190 (Ang2), contributing to the stabilization of the complex. Additional π-π stacking interactions involving His 108 (FGFR2) and Phe 190 (Ang2) underline the importance of aromatic side chains in maintaining the structural integrity of the complex. F A hydrogen bond between Lys 176 (FGFR2, pink) and Glu 274 (AMG386, yellow) reinforces the binding interface. G Another hydrogen bond is observed between Glu 283 (AMG386, yellow) and Gly 200 (Ang2, blue), further enhancing the specificity and stability of the interaction

Article Snippet: Cells were then incubated with 100–1000 ng/ml recombinant Ang2 (expressed with a C-terminal His-tag in CHO cells; Biolegend, No. 753106) or/and 3 or 5 nM FGF1 (R&D Systems, No. 232-FA) for 5 min, and/or 314.8 nM (10 μg/ml) AMG386 (obtained under material CRADA from Amgen).

Techniques: Binding Assay

Journal: Angiogenesis

Article Title: Angiopoietin-2 binds to FGFR2, inhibits FGF-FGFR2 signaling, and delays cutaneous wound healing by inhibiting wound angiogenesis

doi: 10.1007/s10456-025-09988-2

Figure Lengend Snippet: Ang2 reduces FGF1-induced activation of FGFR, Erk1/2, STAT3 and AKT in HUVEC . A HUVEC were incubated in starvation medium only (None), with FGF1 (3 nM), with Ang2 (3 nM) or with Ang2 (3 nM) + FGF1 (3 nM) for the indicated times (min). Cell lysates were immunoblotted with the indicated antibodies. When FGF1 + Ang2 were added to the cells, the incubation time of Ang2 was 5 min and the incubation time of FGF1 was either 1, 2 or 5 minutes (displayed as 5/1, 5/2, 5/5). Representative results of 3 experiments. B HUVEC were incubated in starvation medium only (None), with FGF1 (3 nM), with Ang2 (3 nM) or with Ang2 (3 nM) + FGF1 (3 nM) for the indicated times (min). When Ang2 + FGF1 were added to the cells, the incubation time of Ang2 was 10 min and the incubation time of FGF1 was either 1, 2 or 5 minutes (displayed as 10/1, 10/2, 10/5). Representative of 3 experiments. C HUVEC were incubated in starvation medium only (None), with FGF1 (3 nM), FGF2 (3 nM), Ang2 (3 nM), Ang2 (3 nM) + FGF1 (3 nM) or with Ang2 (3 nM) + FGF2 (3 nM). When Ang2+FGF1 or Ang2+FGF2 were added together to the cells, the incubation time was either 2- or 5-min. Representative of 3 experiments. D Effects of AMG386 on p-Erk1/2 levels in HUVEC activated by FGF1 alone, Ang2 alone, or FGF1 + Ang2. HUVEC were cultured in starvation medium only (none), FGF1 only (5 nM; 5 min), Ang2 only (5 nM; 10 min) or FGF1 (5 nM; 5 min) + Ang2 (5 nM; 10 min). Where indicated, AMG386 (AMG, 315 nM) was added to HUVEC 60 min prior to the addition of FGF1 alone, Ang2 alone or Ang2 + FGF1. Relative band intensity (p-Erk/total Erk) is shown in the bar graph. Representative of 3 experiments

Article Snippet: Cells were then incubated with 100–1000 ng/ml recombinant Ang2 (expressed with a C-terminal His-tag in CHO cells; Biolegend, No. 753106) or/and 3 or 5 nM FGF1 (R&D Systems, No. 232-FA) for 5 min, and/or 314.8 nM (10 μg/ml) AMG386 (obtained under material CRADA from Amgen).

Techniques: Activation Assay, Incubation, Cell Culture

Journal: Angiogenesis

Article Title: Angiopoietin-2 binds to FGFR2, inhibits FGF-FGFR2 signaling, and delays cutaneous wound healing by inhibiting wound angiogenesis

doi: 10.1007/s10456-025-09988-2

Figure Lengend Snippet: Ang2 reduces p-FGFR, p-Erk1/2, p-STAT3 and p-AKT in HEK293T cells. A, B HEK293T cells were incubated in starvation medium only (None), with Ang2 (3 nM), with FGF1 (3 nM), or with Ang2 (3 nM) + FGF1 (3 nM) for the indicated times (min). Cell lysates were immunoblotted with the indicated antibodies. Representative results from 3 experiments. When FGF1 + Ang2 were added to the cells, the incubation time of FGF1 was 5 min and the incubation time of Ang2 is either 1, 2 or 5 minutes (displayed as 5/1, 5/2, 5/5). C HEK293T cells were incubated in starvation medium with FGF2 (3 nM) alone (2 or 5 min), or with Ang2 (3 nM), 5 min. The results show Ang2 + FGF2 for 5 min reduce FGF2-induced Erk1/2 activity (5 min). D Left panel: HEK293T cells were incubated in starvation medium only (None), FGF1 only (5 nM; 5 min), or with Ang2 (5 nM; 5 min) without or with AMG386 (AMG, 5 ng/ml; 1, 2, or 5 min). Right panel: HEK293T cells were incubated with medium only (None), FGF1 only (5 nM; 5 min), or FGF1 (5 nM; 5 min) + Ang2 (5 nM; 5 min), with or without AMG386 (5 ng/ml; 1, 2, or 5 min). Relative band intensity (p-Erk/total Erk) is shown in the bar graph. Representative of 3 experiments

Article Snippet: Cells were then incubated with 100–1000 ng/ml recombinant Ang2 (expressed with a C-terminal His-tag in CHO cells; Biolegend, No. 753106) or/and 3 or 5 nM FGF1 (R&D Systems, No. 232-FA) for 5 min, and/or 314.8 nM (10 μg/ml) AMG386 (obtained under material CRADA from Amgen).

Techniques: Incubation, Activity Assay

Journal: Angiogenesis

Article Title: Angiopoietin-2 binds to FGFR2, inhibits FGF-FGFR2 signaling, and delays cutaneous wound healing by inhibiting wound angiogenesis

doi: 10.1007/s10456-025-09988-2

Figure Lengend Snippet: Ang2 impairs endothelial cell migration induced by FGF1. A Effects of Ang2 (100 ng/ml) and FGF1 (3 ng/ml) individually or together on HUVEC proliferation after 72 hours incubation. Results from 3 H thymidine incorporation are expressed as cpm/culture. Dots reflects results of individual experiments performed in triplicate cultures; experimental means (±SD) are reflected by the bar graphs and error bars. B Ang2 reduces FGF1-induced wound healing in vitro. Images from a representative wound healing assay (of 5 assays) evaluated at 0, 12 and 16 hours (h) after HUVEC wounding. Ang2 (100 ng/ml), FGF1 (10 ng/ml) were added individually or together to the wounded HUVEC monolayers. Quantification of the results from triplicate cultures. The results of % wound closure from individual values (shown as dots) are expressed as mean (±SD), reflected by the error bars. Representative of 5 experiments. C The Ang2 inhibitor, AMG386 (AMG, 5 ng/ml) mitigates inhibition of wound healing by Ang2 (100 ng/ml) in the presence of FGF1 (10 ng/ml). Results of HUVEC wound closure from 4 experiments (evaluated at 12 hours after wounding) are presented as individual dots and means (±SD), reflected by the error bars. D, E Ang2 reduces FGF1-induced HUVEC transmigration. HUVEC (5x10 5 ) were tested in transmigration assays using Transwells (8.0 µm pore size) with or without Ang2 (100 ng/ml) and FGF1 (50 ng/ml). The number of cells migrated to lower surface of the membrane separating the upper from the lower chamber was counted after staining 0.5% crystal violet. Representative images (D) and quantification of results from 3 experiments, each performed in triplicate (E). Significant differences:*P<0.05; ** P<0.01; ***P<0.001 by two-way ANOVA for multiple comparisons with Tukey’s correction

Article Snippet: Cells were then incubated with 100–1000 ng/ml recombinant Ang2 (expressed with a C-terminal His-tag in CHO cells; Biolegend, No. 753106) or/and 3 or 5 nM FGF1 (R&D Systems, No. 232-FA) for 5 min, and/or 314.8 nM (10 μg/ml) AMG386 (obtained under material CRADA from Amgen).

Techniques: Migration, Incubation, In Vitro, Wound Healing Assay, Inhibition, Transmigration Assay, Pore Size, Membrane, Staining