recombinant murine fgf2 3139 fb  (R&D Systems)

Journal: Journal of Cardiovascular Development and Disease

Article Title: FGFR4 Is Required for Concentric Growth of Cardiac Myocytes during Physiologic Cardiac Hypertrophy

doi: 10.3390/jcdd11100320

Figure Lengend Snippet: FGFR4 inhibition abrogates cardiac myocyte hypertrophy induced by serum from fed pythons. Neonatal rat ventricular myocytes (NRVMs) were treated with media containing either vehicle, FGF2 (25 ng/mL), FGF23 (25 ng/mL), or serum from fasted or fed snakes (diluted to 2% of the total media volume), without or in combination with a small molecule inhibitor of FGFR4 (iFGFR4; BLU9931, 10 ng/mL), for 48 h. Compared to vehicle-treated NRVMs (Ctrl) or to NRVMs treated with serum from fasted pythons, serum collected from pythons twelve hours post-feeding (Python 12HPF; #### p < 0.0001) and three days post-feeding (Python 3DPF; #### p < 0.0001) induced a significant increase in myocyte area. When co-treated with iFGFR4 (textured bars), both Python 12HPF (**** p < 0.0001) and Python 3DPF (*** p = 0.0003) did not induce a significant increase in NRVM area when compared to the same treatment without iFGFR4. Python 12HPF ( $$$ p = 0.0003)- and Python 3DPF ( $$ p = 0.0016)-treated NRVMs had significantly increased myocyte area when compared to Python Fasted serum treatments. Treatment with recombinant FGF23 significantly increased myocyte area compared to Ctrl ( #### p < 0.0001), fasted python serum treatments ( $$$$ p < 0.0001), and FGF23 + iFGFR4 treatment (**** p < 0.0001) NRVMs. Treatment with recombinant FGF2 protein served as a positive control for hypertrophy ( ## p = 0.003). Treatment with serum from fasted and two-days-post-feeding water snakes (Water Snake Fasted, Water Snake 2DPF) served as negative controls Each individual color corresponds with the treatment indicated below the bars. # = versus Ctrl; * = versus same treatment + iFGFR4; $ = versus Python Fasted; 150 cells per condition; n = 3–4 independent isolations of NRVMs. All values are shown as mean ± SD.

Article Snippet: We used recombinant murine FGF2 (3139-FB) and FGF23 (2629-FG/CF) proteins from R&D Systems.

Techniques: Inhibition, Recombinant, Positive Control

Journal: bioRxiv

Article Title: KLF4 in smooth muscle cell-derived progenitor cells is essential for angiotensin II-induced cardiac inflammation and fibrosis

doi: 10.1101/2024.06.04.597485

Figure Lengend Snippet: Gli1 -Cre ERT -YFP mice were subjected to Angiotensin II (AngII)-induced cardiac hypertrophy and fibrosis, as described in the Methods section. (A) Masson’s trichrome staining of 4-week AngII or saline treated cardiac tissue; ECM/collagen deposition shown in blue. Representative 20x images of N=4 in each group are shown. (B) Cardiac tissue sections were immunofluorescently stained with anti-GFP antibody and subjected to label-free SHG imaging to visualize the collagen deposition (Red). YFP + cells were imaged (Green) and overlayed to examine their association with ECM/collagen deposition. Representative 40x images of saline (N=10) and AngII-treated (N=12) tissues are shown. ( C ) Cardiac tissue sections were immunofluorescently stained with anti-GFP (Green), anti-αSMA (Red) and DAPI. Representative 60x images of N=6 in each group are shown. (D-I) Cardiac tissue from 2-week AngII- or saline-treated mice were harvested and prepared for scRNA-seq, as described in the Methods section. (D) Uniform Manifold Approximation and Projection (UMAP) visualization is shown. Blue circle highlights the major cluster that is annotated as AdvSca1-SM and fibroblast (SM-Fib) clusters. (E) scRNA-seq data UMAP plot colored by treatment (Saline - blue; AngII - orange). Red circle highlights the AngII-induced shift. (F) YFP transcript positive cells were highlighted in green in the UMAP plot. (G&H) Composition of Saline and AngII-treated samples were visualized for all cells (G) and YFP + cells (H) of the scRNA-seq data. (I) Pathway analysis for genes up-regulated (top) and down-regulated (bottom) by AngII treatment in YFP + AdvSca1-SM cells.

Article Snippet: Sorted cells were plated in gelatin-coated plates with AdvSca1-SM media (α MEM [Gibco, Cat# 32571036]), 10% MSC qualified fetal bovine serum (FBS)(Thermofisher Cat #12662029), 1x Penicillin Streptomycin, 1ng/mL murine basic fibroblast growth factor (R&D systems 3139-FB), and 5ng/mL murine epidermal growth factor (R&D systems 2028-EG) at the density of 20,000/cm 2 .

Techniques: Staining, Saline, Imaging

Journal: Genome biology

Article Title: YY1 lactylation in microglia promotes angiogenesis through transcription activation-mediated upregulation of FGF2.

doi: 10.1186/s13059-023-02931-y

Figure Lengend Snippet: Fig. 5 YY1 lactylation contributes to angiogenesis by regulating FGF2 expression. a The mRNA expression of VEGFA, FGF2, MMP2, MMP9, and ANGPTL6 in the retinal tissue of normal control (Normal), OIR (DMSO), and OIR (PLX3397) mice (n = 3 per group). b The mRNA expression of VEGFA, FGF2, MMP2, MMP9, and ANGPTL6 in HMC3 cells in the three groups subjected to hypoxia for 0, 12, and 24 h (n = 3 per group). c The protein expression of FGF2 in the retinas of normal control (Normal), OIR (DMSO), and OIR (PLX3397) mice (n = 3 per group). d The protein expression of FGF2 in the HMC3 cells subjected to hypoxia for 0 and 24 h (n = 3 per group). e Representative images of FGF2 co-stained with microglia (Iba1) in retina of OIR mice (OIR P17) and control mice (Normal P17), with quantification of FGF2 intensity (n = the number of Iba1+ cells); scale bar, 20 μm. f The protein expression of FGF2 in HMC3 cells exposed to hypoxia 24 h (Hypoxia Control), hypoxia for 24 h + YY1 WT transfection (Hypoxia + WT), and hypoxia for 24 h + YY1 K183R transfection (Hypoxia + K183R) (n = 3 per group). g ChIP-qPCR analysis of the indicated promoters was performed by using of YY1 antibody in HMC3 cells treated with normoxia and hypoxia (n = 3 per group). h ChIP-qPCR analysis of the indicated promoters was performed by using of YY1 antibody in HMC3 cells overexpressing WT and K183R YY1 (n = 3 per group). i The luciferase activity of the FGF2 promoter driven reporter vector was measured in response to YY1 WT or K183R cotransfection under hypoxia (n = 6 per group). NS, non-significance; *p <0.05; **p < 0.01; ***p < 0.001

Article Snippet: One microliter of recombinant murine FGF2 protein (100 ng/ ml, R&D Systems, 3139- FB) was administered by intravitreal injection to the eyes at P14.

Techniques: Expressing, Control, Staining, Transfection, ChIP-qPCR, Luciferase, Activity Assay, Plasmid Preparation, Cotransfection

Journal: Genome biology

Article Title: YY1 lactylation in microglia promotes angiogenesis through transcription activation-mediated upregulation of FGF2.

doi: 10.1186/s13059-023-02931-y

Figure Lengend Snippet: Fig. 6 p300 affects the lactylation of YY1 and then regulates angiogenesis in vitro. a Quantification of p300 in the HMC3 cells under normoxia or hypoxia for 24 h (n = 3 per group). b Co-IP analysis of YY1 and p300. c Colocalization of YY1 and p300 by double-label immunofluorescence. d The Pan-Kla level in the HMC3 cells overexpressing p300 (n = 3 per group). e Overexpression of p300 increased YY1 lactylation. Lactylation of YY1 in control group, or oep300-WT group was detected by YY1-Kla antibodies (n = 3 per group). f The expression of FGF2 and p300 in the HMC3 cells overexpressing p300 (n = 3 per group). g Quantification of FGF2 and Pan-Kla in the HMC3 cells treated with 0, 5, 10, and 20 μM A-485 under hypoxia for 24 h (n = 3 per group). h Applying A-485 decreased YY1 lactylation. Lactylation of YY1 with 0 μM A-485, or 20 μM A-485 was detected by YY1-Kla antibodies (n = 3 per group). *p < 0.05; **p < 0.01; ***p < 0.001

Article Snippet: One microliter of recombinant murine FGF2 protein (100 ng/ ml, R&D Systems, 3139- FB) was administered by intravitreal injection to the eyes at P14.

Techniques: In Vitro, Co-Immunoprecipitation Assay, Immunofluorescence, Over Expression, Control, Expressing

Journal: Genome biology

Article Title: YY1 lactylation in microglia promotes angiogenesis through transcription activation-mediated upregulation of FGF2.

doi: 10.1186/s13059-023-02931-y

Figure Lengend Snippet: Fig. 7 Inhibiting p300 reduces the lactylation of YY1 and suppresses angiogenesis in OIR. a p300, YY1, and YY1-Kla in the retinas of normal control (Normal P17) and OIR (OIR P17) mice (n = 3 per group). b Representative images of YY1-Kla co-stained with microglia (Iba1) in retina of OIR mice (OIR P17) and control mice (Normal P17), with quantification of YY1-Kla intensity (n = the number of Iba1+ cells); scale bar, 20 μm. c Quantification of YY1-Kla and FGF2 in the retinas of OIR mice (OIR P17) treated with A-485 (200 μM, 1 μl/eye) and DMSO (0.1%) were analyzed by Western blotting (n = 3 per group). d Representative images of YY1-Kla co-stained with microglia (Iba1) in retina of OIR mice treated with A-485 (200 μM, 1 μl/eye) and OIR mice treated with DMSO (0.1%), with quantification of YY1-Kla intensity (n = the number of Iba1+ cells); scale bar, 20 μm. e Confocal images of CD31-stained retinal flat mounts in OIR P17 mice treated with A-485 (200 μM, 1 μl/eye) and DMSO (0.1%). The relative proportion of neovascularization in the retina was calculated and measured by ImageJ (n = 4 mice per group); scale bar, 1000 μm. f Representative images of FGF2 co-stained with microglia (Iba1) in retina of OIR mice (OIR P17) treated with A-485 (200 μM, 1 μl/eye) and DMSO (0.1%), with quantification of FGF2 intensity (n = the number of Iba1+ cells); scale bar, 20 μm. g Confocal images of CD31 and Iba1-stained retinal flat mounts in OIR P17 mice treated with A-485 (200 μM, 1 μl/eye) and DMSO (0.1%); the yellow arrow denotes the resting-state of microglia, and the green arrow denotes the activated state of microglia; scale bar, 50 μm. *p < 0.05; ***p < 0.001

Article Snippet: One microliter of recombinant murine FGF2 protein (100 ng/ ml, R&D Systems, 3139- FB) was administered by intravitreal injection to the eyes at P14.

Techniques: Control, Staining, Western Blot

Journal: Genome biology

Article Title: YY1 lactylation in microglia promotes angiogenesis through transcription activation-mediated upregulation of FGF2.

doi: 10.1186/s13059-023-02931-y

Figure Lengend Snippet: Fig. 8 Schematic diagram illustrates the molecular mechanism how YY1 lactylation in microglia contributes to angiogenesis. YY1 lactylation in microglia is upregulated in response to hypoxia. Hyperlactylation of YY1 promotes FGF2 transcription and then contributes to neovascularization

Article Snippet: One microliter of recombinant murine FGF2 protein (100 ng/ ml, R&D Systems, 3139- FB) was administered by intravitreal injection to the eyes at P14.

Techniques:

Journal: Nature Communications

Article Title: Menin directs regionalized decidual transformation through epigenetically setting PTX3 to balance FGF and BMP signaling

doi: 10.1038/s41467-022-28657-2

Figure Lengend Snippet: a GSEA plot evaluating the changes in the indicated gene signatures of response to FGF ( n = 46) and response to BMP ( n = 57) in Men1 f/f uteri compared with Men1 d/d uteri. NES, normalized enrichment score. b Heatmap of differentially expressed genes between Men1 f/f and Men1 d/d uteri on day 8. RPKM of each gene from different samples was normalized to Z-score. c , d Quantitative real-time PCR analysis of Bmp2 , Id2 , Fgfr1 , and Fgf2 mRNA levels in Men1 f/f and Men1 d/d uteri on day 8. The values are normalized to Gapdh and indicated as the mean ± SEM ( n = 3 biologically independent samples). Two-tailed unpaired Student’s t -test, * p = 0.02 ( Fgfr1 ), ** p = 0.007 ( Bmp2 ), * p = 0.046 ( Id2 ). ns not significant. e Immunoblotting analysis of BMP2 and p-SMAD1/5/8 in Men1 f/f and Men1 d/d uteri. Β-Actin and t-SMAD1/5/8 were used as loading controls. f In situ hybridization of Bmp2 and Fgfr1 in Men1 f/f and Men1 d/d uteri on day 8. AM antimesometrial pole. Scale bar: 200 μm. g Immunohistochemical staining of phosphorylated ERK1/2 and total ERK1/2 in Men1 f/f and Men1 d/d uteri on day 8. Scale bar: 200 μm.

Article Snippet: The media was changed every 48 h. For the treatment of primary uterine stromal cells, MI-503 (2 μM; MedChemExpress), recombinant murine FGF2 (PeproTech, 20 ng/ml), recombinant mouse PTX3 (R&D systems, 100 ng/ml) and MEK inhibitor PD0325901 (Selleck, 5 μM) were used.

Techniques: Real-time Polymerase Chain Reaction, Two Tailed Test, Western Blot, In Situ Hybridization, Immunohistochemical staining, Staining

Journal: Nature Communications

Article Title: Menin directs regionalized decidual transformation through epigenetically setting PTX3 to balance FGF and BMP signaling

doi: 10.1038/s41467-022-28657-2

Figure Lengend Snippet: a Genome browser view of normalized Menin and H3K4me3 ChIP-seq signals at Ptx3 promoter locus and RNA-seq tracks for Ptx3 in Men1 f/f and Men1 d/d decidual tissues. b Quantitative real-time PCR analysis of Ptx3 in Men1 f/f and Men1 d/d uteri on day 8. The values are normalized to Gapdh and indicated as the mean ± SEM ( n = 3 biologically independent samples). Two-tailed unpaired Student’s t -test, **** p = 1.07e-5. c Quantitative ChIP analysis of Menin at Ptx3 promoter in uterine decidual tissue. Data represent the mean ± SEM. Two-tailed unpaired Student’s t -test, *** p = 0.0009. d Quantitative ChIP analysis of H3K4me3 at Ptx3 promoter in Men1 f/f and Men1 d/d uterine decidual tissue. Data represent the mean ± SEM ( n = 3 biologically independent samples). Two-tailed unpaired Student’s t -test, ** p = 0.0025. e In situ hybridization of Ptx3 in Men1 f/f and Men1 d/d uteri on day 8. Scale bar: 200 μm. f Immunoblotting analysis of the phosphorylation of ERK1/2 in mouse primary endometrial stromal cells treatment with FGF2 (20 ng/ml) in the absence or presence of PTX3 (100 ng/ml). Total ERK1/2 and β-Actin were used as loading controls. g Quantitative real-time PCR analysis of Ptx3 in primary mESC treated with DMSO or MI-503(2 μM) in vitro. The values are normalized to Gapdh and indicated as the mean ± SEM ( n = 3 biologically independent samples). Two-tailed unpaired Student’s t -test, ** p = 0.0053. h Immunoblotting analysis of Menin, BMP2, and phosphorylated ERK1/2 after the addition of PTX3 (100 ng/ml) or PD0325901 (5 μM) in mESC treatment with MI-503 (2 μM) in the presence of FGF2 (20 ng/ml). Total ERK1/2 and β-Actin were used as loading controls.

Article Snippet: The media was changed every 48 h. For the treatment of primary uterine stromal cells, MI-503 (2 μM; MedChemExpress), recombinant murine FGF2 (PeproTech, 20 ng/ml), recombinant mouse PTX3 (R&D systems, 100 ng/ml) and MEK inhibitor PD0325901 (Selleck, 5 μM) were used.

Techniques: ChIP-sequencing, RNA Sequencing, Real-time Polymerase Chain Reaction, Two Tailed Test, In Situ Hybridization, Western Blot, Phospho-proteomics, In Vitro

Journal: Nature Communications

Article Title: Menin directs regionalized decidual transformation through epigenetically setting PTX3 to balance FGF and BMP signaling

doi: 10.1038/s41467-022-28657-2

Figure Lengend Snippet: a Uterine deletion of Men1 leads to abnormal polyploidization of decidual cells in the SDZ, accompanied by impeded maturation of uNK cells, increased inflammatory response, and shallow trophoblast invasion. b Menin positively regulates the expression of Ptx3 in an H3K4me3 dependent manner. PTX3, trapping FGF2 from undifferentiated stromal cells, ensures the expression of BMP2 in the SDZ by inhibiting the activation of FGF2-ERK1/2.

Article Snippet: The media was changed every 48 h. For the treatment of primary uterine stromal cells, MI-503 (2 μM; MedChemExpress), recombinant murine FGF2 (PeproTech, 20 ng/ml), recombinant mouse PTX3 (R&D systems, 100 ng/ml) and MEK inhibitor PD0325901 (Selleck, 5 μM) were used.

Techniques: Expressing, Activation Assay