Journal: The Journal of Infectious Diseases

Article Title: FGF8 Protects Against Polymicrobial Sepsis by Enhancing the Host's Anti-infective Immunity

doi: 10.1093/infdis/jiae559

Figure Lengend Snippet: Kinetics of FGF8 production in polymicrobial sepsis. A , Male C57BL/6 mice (n = 5 per group) were subjected to sham or CLP using a 24-gauge needle. PLF, serum, lung, spleen and kidney were collected at the indicated times (6, 24, 48 hours) after CLP. FGF8 concentrations were measured by ELISA. B , Representative fluorescence images of FGF8 expression in kidney, spleen, and lung after CLP. Scale bar = 50 μm. Quantitative results are shown (n = 3 per group). C , FGF8 concentrations in serum and PLF of TLR2 −/− , TLR4 −/− , TLR2/4 −/− , and WT mice 24 hours (n = 3–4 per group) after CLP. D , Supernatant of heat-killed Pseudomonas aeruginosa (MOI = 1:100) challenged macrophages was collected at the indicated times (6, 12, 24 hours). FGF8 levels were measured by ELISA (n = 4 per group). A–D , Data are representative of 3 independent experiments; Kruskal-Wallis test followed by Dunn multiple comparisons posttest. * P < .05, ** P < .01, *** P < .001, **** P < .0001 compared within 2 groups. Abbreviations: CLP, cecal ligation and puncture; Ctrl, control; ELISA, enzyme-linked immunosorbent assay; FGF, fibroblast growth factor; MOI, multiplicity of infection; ns, not significant; PLF, peritoneal lavage fluid; TLR, Toll-like receptor; WT, wild type.

Article Snippet: FGF8 blockade was performed by IP administration of 25 μg/kg of mouse monoclonal anti-FGF8 antibody (MAB323, R&D Systems) immediately after CLP.

Techniques: Enzyme-linked Immunosorbent Assay, Fluorescence, Expressing, Ligation, Control, Infection

Journal: The Journal of Infectious Diseases

Article Title: FGF8 Protects Against Polymicrobial Sepsis by Enhancing the Host's Anti-infective Immunity

doi: 10.1093/infdis/jiae559

Figure Lengend Snippet: Inhibition of endogenous FGF8 exacerbated polymicrobial sepsis. A , Survival of septic mice (n = 20 per group) with or without FGF8 neutralization after CLP using a 24-gauge needle. B , Dilutions of PLF, blood, liver, lung, and spleen tissues were obtained from septic mice (n = 10–11 per group) treated with or without monoclonal anti-FGF8 antibody at 24 hours after CLP; samples were cultured on blood agar plates and the numbers of bacterial colonies were then determined. C , Representative examples of hematoxylin and eosin-stained tissues as indicated from mice (n = 5 per group) treated or not treated with monoclonal anti-FGF8 antibody at 24 hours after CLP. The pathology scores are shown on the right side of histological images. D , Serological markers of organ injury including ALT, AST, LDH, creatinine, and urea in septic mice (n = 11–12 per group) treated with or without monoclonal anti-FGF8 antibody at 24 hours after CLP. Each dot represents an individual mouse. B–D , Nonparametric Mann-Whitney U test; ( A ) Kaplan-Meier analysis followed by log-rank test. * P < .05, ** P < .01, *** P < .001 compared within 2 groups. Abbreviations: ALT, alanine transaminase; AST, aspartate transaminase; CLP, cecal ligation and puncture; FGF, fibroblast growth factor; IgG, immunoglobulin G; LDH, lactate dehydrogenase; ns, not significant; PLF, peritoneal lavage fluid.

Article Snippet: FGF8 blockade was performed by IP administration of 25 μg/kg of mouse monoclonal anti-FGF8 antibody (MAB323, R&D Systems) immediately after CLP.

Techniques: Inhibition, Neutralization, Cell Culture, Staining, MANN-WHITNEY, Ligation

Journal: The Journal of Infectious Diseases

Article Title: FGF8 Protects Against Polymicrobial Sepsis by Enhancing the Host's Anti-infective Immunity

doi: 10.1093/infdis/jiae559

Figure Lengend Snippet: FGF8 treatment improves outcomes in a murine sepsis model. A , Different levels of rFGF8 (0, 5, 12.5 μg/kg) were injected intraperitoneally into mice (n = 20 per group) immediately after CLP and survival was monitored for 14 days. B , Dilutions of blood, lungs, PLF, and spleens obtained from septic mice (n = 9–10 per group) treated with or without rFGF8 (12.5 μg/kg) 48 hours after CLP. C , Representative examples of hematoxylin and eosin-stained lung, liver, spleen, and kidney tissues from CLP mice treated with or without rFGF8 (12.5 μg/kg) after CLP. Scale bars = 400 μm. Histological scores (n = 5 per group) are shown. D , Serological markers of organ injury in septic mice (n = 13 per group) treated with or without rFGF8 (12.5 μg/kg) at 48 hours after CLP. B–D , Data are representatives of 3 independent experiments; nonparametric Mann-Whitney U test; ( A ) Kaplan-Meier analysis followed by log-rank test. * P < .05, ** P < .01 compared within 2 groups. Abbreviations: ALT, alanine transaminase; AST, aspartate transaminase; CLP, cecal ligation and puncture; FGF, fibroblast growth factor; LDH, lactate dehydrogenase; ns, not significant; PBS, phosphate-buffered saline; PLF, peritoneal lavage fluid.

Article Snippet: FGF8 blockade was performed by IP administration of 25 μg/kg of mouse monoclonal anti-FGF8 antibody (MAB323, R&D Systems) immediately after CLP.

Techniques: Injection, Staining, MANN-WHITNEY, Ligation, Saline

Journal: The Journal of Infectious Diseases

Article Title: FGF8 Protects Against Polymicrobial Sepsis by Enhancing the Host's Anti-infective Immunity

doi: 10.1093/infdis/jiae559

Figure Lengend Snippet: FGF8 directly enhances bacterial phagocytosis and killing by macrophages. A , Peritoneal macrophages (1 × 10 6 cells) were stimulated with or without rFGF8 (200 ng/mL) for 6 hours and then challenged with FITC-labeled Pseudomonas aeruginosa (MOI = 1:100) for 30 minutes at 37°C. Representative images from 3 independent experiments are shown. Dot plot depicts macrophage phagocytosis levels (n = 5 per group). B , Bacterial killing of P. aeruginosa in peritoneal macrophages (5 × 10 5 cells) treated with PBS or the indicated doses of rFGF8. Dot plot depicts macrophage killing (n = 4 per group). C , Mortality of rFGF8-treated (12.5 μg/kg) septic mice in the presence or absence of macrophage depletion after CLP (n = 10 per group). D , Bacterial loads in PLF and blood of rFGF8-treated (12.5 μg/kg) septic mice in the presence or absence of macrophage depletion after CLP (n = 5 per group). E , Survival after transfer of rFGF8- or PBS-treated peritoneal macrophages in mice (n = 12 per group) after CLP. A, B, and D , Data are representatives of 3 independent experiments; ( A ) nonparametric Mann-Whitney U test; ( B and D ) Kruskal-Wallis test followed by Dunn multiple comparisons posttest; ( C and E ) Kaplan-Meier analysis followed by log-rank test. * P < .05, ** P < .01, *** P < .001, **** P < .0001 compared within 2 groups. Abbreviations: CLP, cecal ligation and puncture; CFU, colony-forming unit; DAPI, 4′,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate; MOI, multiplicity of infection; ns, not significant; PBS, phosphate-buffered saline; rFGF, recombinant fibroblast growth factor; TRITC, tetraethyl rhodamine isothiocyanate.

Article Snippet: FGF8 blockade was performed by IP administration of 25 μg/kg of mouse monoclonal anti-FGF8 antibody (MAB323, R&D Systems) immediately after CLP.

Techniques: Labeling, MANN-WHITNEY, Ligation, Infection, Saline, Recombinant

Journal: The Journal of Infectious Diseases

Article Title: FGF8 Protects Against Polymicrobial Sepsis by Enhancing the Host's Anti-infective Immunity

doi: 10.1093/infdis/jiae559

Figure Lengend Snippet: FGFR1 plays a critical role in FGF8-induced protection against experimental sepsis. A , Representative confocal images of the colocalization of FGF8 (Cy3) and FGFR (FITC) in peritoneal macrophages treated with rFGF8 (200 ng/mL). Scale bar = 20 μm. B , Peritoneal macrophages were pretreated with or without rFGF8 (200 ng/mL) followed by incubation with or without heat-inactivated Pseudomonas aeruginosa. Representative fluorescence images of phospho-FGFR1 expression are shown. Scale bar = 25 μm. C , Peritoneal macrophages (n = 4 per group) were pretreated with or without the FGFR1 inhibitor PD173074 (100 nM) for 1 hour followed by incubation with or without rFGF8 (200 ng/mL). In vitro bacterial phagocytosis and killing of P. aeruginosa . D , Mortality after blocking FGFR1 with PD173074 (1 mg/kg) and subsequent treatment with rFGF8 (12.5 μg/kg) or PBS control after CLP (n = 15 per group). Except for survival ( D ), data are presented as means and are representative of 3 independent experiments; ( C ) Kruskal-Wallis test followed by Dunn multiple comparisons posttest; ( D ) Kaplan-Meier analysis followed by log-rank test. * P < .05, *** P < .001, **** P < .0001 compared within 2 groups. Abbreviations: CLP, cecal ligation and puncture; FGFR, FGF receptor; ns, not significant; rFGF, recombinant fibroblast growth factor; FITC, Fluorescein Isothiocyanate; Cy3, Cyanine 3.

Article Snippet: FGF8 blockade was performed by IP administration of 25 μg/kg of mouse monoclonal anti-FGF8 antibody (MAB323, R&D Systems) immediately after CLP.

Techniques: Incubation, Fluorescence, Expressing, In Vitro, Blocking Assay, Control, Ligation, Recombinant

Journal: The Journal of Infectious Diseases

Article Title: FGF8 Protects Against Polymicrobial Sepsis by Enhancing the Host's Anti-infective Immunity

doi: 10.1093/infdis/jiae559

Figure Lengend Snippet: FGF8-enhanced antibacterial functions of macrophages are regulated by ERK1/2 signaling pathways. A , Peritoneal murine macrophages were treated with or without rFGF8 (200 ng/mL) for the indicated times and examined for the presence of phosphorylated ERK1/2, P38, STAT1, and Akt. Three independent experiments were performed with comparable results and representative blots are shown. B , Effects of rFGF8 on the activation of MAPK, STAT, PI3 K signaling pathways in murine macrophages. Peritoneal macrophages were treated with or without rFGF8 (200 ng/mL) and then incubated with heat-inactivated Pseudomonas aeruginosa . Total cellular proteins were extracted from murine macrophages for the detection of phosphorylated ERK1/2, P38, STAT1, and Akt with the indicated antibodies by western blot analysis. Experiments were performed in 3 independent experiments with consistent results and representative blots are shown. Peritoneal macrophages (n = 4 per group) were pretreated with the ERK1/2 inhibitor U0126 (20 μM) for 1 hour followed by incubation with or without rFGF8 (200 ng/mL). C , Analysis of in vitro bacterial phagocytosis and killing of P. aeruginosa . D , Mortality after blocking ERK1/2 signaling pathway with U0126 (10 mg/kg) and subsequent treatment with rFGF8 (12.5 μg/kg) or PBS control after CLP (n = 20 per group). Except for survival rate ( D ), data are presented as means and are representative of 3 independent experiments; ( C ) Kruskal-Wallis test followed by Dunn multiple comparisons posttest; ( D ) Kaplan-Meier analysis followed by log-rank test. * P < .05, ** P < .01, **** P < .0001 compared within 2 groups. Abbreviations: CFU, colony-forming unit; CLP, cecal ligation and puncture; DMSO, dimethyl sulfoxide; ns, not significant; PBS, phosphate-buffered saline; rFGF, recombinant fibroblast growth factor.

Article Snippet: FGF8 blockade was performed by IP administration of 25 μg/kg of mouse monoclonal anti-FGF8 antibody (MAB323, R&D Systems) immediately after CLP.

Techniques: Protein-Protein interactions, Activation Assay, Incubation, Western Blot, In Vitro, Blocking Assay, Control, Ligation, Saline, Recombinant

Journal: The Journal of Infectious Diseases

Article Title: FGF8 Protects Against Polymicrobial Sepsis by Enhancing the Host's Anti-infective Immunity

doi: 10.1093/infdis/jiae559

Figure Lengend Snippet: FGF8 is a candidate biomarker for sepsis. A , Levels of FGF8 at admission measured by ELISA in serum samples collected from 73 adult patients with sepsis, 96 child patients with sepsis, and corresponding healthy controls. B , Receiver operating characteristic analysis of FGF8 for diagnosis of sepsis (AUC = 0.89 for adult and AUC = 0.81 for children). C , Comparison of serum FGF8 levels between male and female patients with sepsis and healthy controls. D , Comparison of serum FGF8 levels between adults and children in healthy controls and patients with sepsis. A , C , and D , Nonparametric Mann-Whitney U test. **** P < .0001 compared within 2 groups. Abbreviations: AUC, area under the curve; CI, confidence interval; ELISA, enzyme-linked immunosorbent assay; FGF, fibroblast growth factor; ns, not significant.

Article Snippet: FGF8 blockade was performed by IP administration of 25 μg/kg of mouse monoclonal anti-FGF8 antibody (MAB323, R&D Systems) immediately after CLP.

Techniques: Biomarker Discovery, Enzyme-linked Immunosorbent Assay, Comparison, MANN-WHITNEY

Journal: Development (Cambridge, England)

Article Title: Delta-like ligand 4-mediated Notch signaling controls proliferation of second heart field progenitor cells by regulating Fgf8 expression

doi: 10.1242/dev.185249

Figure Lengend Snippet: Dll4 expression in SHF cells is required to maintain expression of key SHF-related proteins. (A-B‴) Transverse sections were evaluated for Mef2c and Fgf8 transcript expression at E9 in control (A) and Mef2c-AHF-Cre,Dll4F/F mutant (B) by RNAscope. Higher magnification of the boxed areas in A and B are shown as Mef2c expression (A′,B′), Fgf8 expression (A″,B″) and merged images (A‴,B‴) to demonstrate the reduced expression of Fgf8 transcripts in the mutants compared with the controls in the pharyngeal mesoderm (PM). (C-D‴) Similarly, transverse sections were evaluated for Mef2c and Fgf10 transcript expression at E9 in control (C) and Mef2c-AHF-Cre,Dll4F/F mutant (D). Higher magnification of the boxed areas in C and D are shown as Mef2c expression (C′,D′), Fgf10 expression (C″,D″) and merged images (C‴,D‴) to demonstrate that the PM in mutants has decreased expression of Fgf10 transcripts. (E-F‴) Transverse sections of control (E) and Mef2c-AHF-Cre,Dll4F/F mutant (F) E9.5 embryos were co-stained for Islet1 and Fgf8 protein expression. Higher magnification of the boxed areas in E and F are shown as Islet1 expression (E′,F′), Fgf8 expression (E″,F″) and merged images (E‴,F‴), showing reduced expression of Fgf8 in the SHF region. (G-H″) Transverse sections of control (G) and Mef2c-AHF-Cre,Dll4F/F mutant (H) E11.5 embryos were stained for Hand2 protein expression. Higher magnification of the boxed areas in G and H show the RV and LV in control (G′,G″) and mutant (H′,H″) embryos. Hand2 expression is lost in the mutant RV compared with controls. There is no change in the low basal level expression seen in LV. Scale bars: 50 µm (E′-F‴); 100 µm (A′-D‴,E,F,G′-H″); 250 µm (A-D,G-H).

Article Snippet: Thoracic organs were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin and varying doses of recombinant Fgf8 (Novus Biologicals, NBP2-35033) for 8 h. The organs were then cryoembedded.

Techniques: Expressing, Control, Mutagenesis, RNAscope, Staining

Journal: Development (Cambridge, England)

Article Title: Delta-like ligand 4-mediated Notch signaling controls proliferation of second heart field progenitor cells by regulating Fgf8 expression

doi: 10.1242/dev.185249

Figure Lengend Snippet: Dll4-mediated notch signaling regulates Fgf8 expression in SHF. (A) Schematic of the mouse chromosome 10 around the region of the Fgf8 gene (E, exon). Putative RBPjk binding sites are indicated with asterisks. Constructs cloned for luciferase assay are shown as black boxes. Promoter 3 and Enhancer 2 were used as negative controls. (B) 293T cells were treated with DAPT to quench basal Notch activity. They were then transfected with various luciferase expression vectors with (empty bars) or without (solid bars) the NICD expression vector. Luciferase activity was measured in triplicate wells (mean±s.e.m.) 24 h later with eight experimental repeats. (C) The experiment was then repeated in triplicate after mutating the putative RBPjk binding site of Promoter 1. Mutation of putative binding sites led to loss of luciferase activity. (D-F″) Thoracic regions were dissected in control (D-D″) and Mef2c-AHF-Cre,Dll4F/F mutant (E-F″) embryos at E9.5 and cultured in vitro. Mutant organs were cultured with (F-F″) or without (E-E″) exogenous recombinant Fgf8. Sections were then co-stained for Islet1 and pHH3 expression to study SHF proliferation. Representative images are shown as Islet1 expression (D′,E′,F′), pHH3 expression (D″,E″,F″) and merged images (D,E,F). (G) Double-positive cells were counted in multiple fields (23 untreated control, 23 Fgf8 100 ng/µl control, 40 Fgf8 500 ng/µl control, 37 Fgf8-untreated mutant, 7 Fgf8 100 ng/µL mutant and 14 Fgf8 500 ng/µl mutant sections; mean±s.e.m.) showing a significant reduction in SHF proliferation in mutant organs compared with control (P<0.0001 between Fgf8-untreated control and mutant, P>0.05 between Fgf8-untreated controls and Fgf8-treated mutants by two-tailed t-tests). For quantification purposes, the boxed regions in D′, E′ and F′ were used as the area occupied by SHF progenitor cells. Exogenous Fgf8 supplementation had no significant impact on control embryos, but fully rescued proliferation defects seen in mutant embryos. (H-L′) Compound heterozygotes were analyzed by H&E staining of transverse sections of E14.5 embryos to demonstrate genetic synergy between Dll4-mediated Notch and Fgf8 signaling in SHF maturation. Cre-negative control embryos showed fully septated ventricles (H) and an aortic valve normally aligned over the left ventricle (H′). Heterozygous knockdown of Dll4 driven by Mef2c-AHF-Cre (Mef2c-Cre,Dll4F/wt) also demonstrated a normal phenotype (I,I′). Heterozygous knockdown of Fgf8 driven by Mef2c-AHF-Cre (Mef2c-Cre,Fgf8F/wt) showed a low incomplete penetrance of cardiac defects. The majority of the embryos showed a normal phenotype (J,J′). A shallow VSD (arrow in K) and a slightly mal-aligned aorta mildly over-riding the ventricular septum (arrowhead in K′) was seen in 14% of the embryos. Double heterozygous knockdown of Dll4 and Fgf8 driven by Mef2c-AHF-Cre (Mef2c-Cre,Dll4F/wt,Fgf8F/wt) showed high penetrance of DORV, with 83% of the embryos studied showing VSD (arrow in L) and a prominent over-riding of aorta with greater than 50% aorta arising from the RV (arrowhead in L′). (M) Table indicates number and phenotypes of embryos recovered amongst the different genotypes shown. The number of embryos recovered, the percentage recovery and the expected percentage recovery are based on Mendelian inheritance. Scale bars: 50 µm (D-F″); 300 µm (H-L′).

Article Snippet: Thoracic organs were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin and varying doses of recombinant Fgf8 (Novus Biologicals, NBP2-35033) for 8 h. The organs were then cryoembedded.

Techniques: Expressing, Binding Assay, Construct, Clone Assay, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Mutagenesis, Control, Cell Culture, In Vitro, Recombinant, Staining, Two Tailed Test, Negative Control, Knockdown

Journal: Human Molecular Genetics

Article Title: Loss-of-function mutations in FGF8 can be independent risk factors for holoprosencephaly

doi: 10.1093/hmg/ddy106

Figure Lengend Snippet: Clinical and molecular findings of genetic variants of the human FGF8 gene

Article Snippet: Expression constructs The FGF8 cDNA, transcript variant e-form (NM_033164.3) has been described ( 29 ) and f-form (NM_033163.3) was purchased from (OriGene Technologies, Rockville, MD).

Techniques: Functional Assay, Negative Control, Binding Assay

Journal: Human Molecular Genetics

Article Title: Loss-of-function mutations in FGF8 can be independent risk factors for holoprosencephaly

doi: 10.1093/hmg/ddy106

Figure Lengend Snippet: Alternative splice forms FGF8e and FGF8f. (A) The schematic genomic organization of the FGF8 locus (exons not drawn to scale). The locus includes six exons that can be alternatively spliced to form four isoforms (FGF8 a, b, e and f). The two longest isoforms (e and f) differ by the alternative use of splice acceptor sites located 33 bp apart in the 5′ region of exon 1d. The 11 codons (indicated in yellow) account for a key additional contact residue that leads to differential binding to FGFR receptors. Variable use of exon 1c (not shown) accounts for two additional isoforms (FGF8a and FGF8b). (B) Biological response of FGF8e versus FGF8f. The biological response has been measured by injecting increasing amounts of synthetic FGF8 mRNA of both forms in separate experiments and monitoring the phenotypic severity by scoring clutches of embryos. Note the dose–response is non-linear. The FGF8e form was the most sensitive to small changes in mRNA dose. A normal phenotype is shown in yellow, blue is for dorsalized embryos and red is for dead embryos. For each tested dose, the phenotypic responses are compared between the two isoforms in all three categories (yellow, blue and red). Error bars represent SEM. An asterix is used (*) to indicated that a given category (denoted by color) reaches a minimal level of significance (P < 0.05) between the two isoforms tested at the same dose. (C) A group image of dusp6 gene expression as a biomarker sensitive to FGF8 dose. Representative comparisons are shown between FGF8e and FGF8f followed by whole mount in situ hybridization at 8 h after fertilization. Quantification data in this study are described in Supplementary Material, Table S2.

Article Snippet: Expression constructs The FGF8 cDNA, transcript variant e-form (NM_033164.3) has been described ( 29 ) and f-form (NM_033163.3) was purchased from (OriGene Technologies, Rockville, MD).

Techniques: Residue, Binding Assay, Gene Expression, Biomarker Discovery, In Situ Hybridization

Journal: Human Molecular Genetics

Article Title: Loss-of-function mutations in FGF8 can be independent risk factors for holoprosencephaly

doi: 10.1093/hmg/ddy106

Figure Lengend Snippet: Functional analysis of FGF8 variants in zebrafish. (A) Schematic representative of amino acid positions of variants and controls as measured by site-directed changes on the FGF8e isoform backbone (see proper names in Table 1). FGF receptor binding domain (green) and heparin binding domain (black) represent the FGF core structural elements common to all ligands in the FGF family (CDD: 238015). (B) Representative illustration of the FGF8e overexpression phenotypes at 24 hpf in zebrafish. Two clear phenotypic classes of embryos are observed in lateral (full image) and ventral view (inset image). The yellow color represents normal embryos and red is for dorsalized. (C) Results of FGF8 mRNA injection. A 0.4 pg of FGF8e mRNA has been introduced into fertilized one-cell stage embryos and their biological response was measured by visual scoring of their phenotype at 24 hpf. The yellow color represents normal, red is for dead and blue for dorsalized by RNA injection. Normal phenotype was only observed in FGF8 T108M and FGF8 15bp del, which is consistent with a complete loss of function. Compared with FGF8 wt, FGF8 T122M, FGF8 V146F, FGF8 N155S and FGF8 R178H all present with a hypomorphic response. Error bars represent SEM, * indicates P < 0.05, *** indicates P < 0.001 and **** indicates P < 10−8 when compared with FGF8 wt based on Student t-test. When appropriate, each category (by color) was independently compared with the expectation based on the wt response and given a statistical result. (D) Whole mount in situ hybridization of dusp6 at 8 h. wt FGF8e is expected to produce a hyperactive induction of dusp6 (FGF8e wt, 18bp ins, p.R44W, p.E56Q, p.V146I). Complete loss-of-function alleles are expected to stain normally (right panel; gfp control, FGF8e p.T108M and FGF8e 15bp del) while hypomorphic activity is somewhere between these extremes (middle panel; FGF8e V146F, N155S, R178H). (E) Western blot of FGF8 variants within the receptor binding domain region that demonstrate variable bioactivity. All constructs were tagged with Flag and transfected into Cos7 cell line and β-actin was used as loading control. Note that p.N155S migrates slightly faster (Fig. 3). Quantification data in this study are described in Supplementary Material, Table S2.

Article Snippet: Expression constructs The FGF8 cDNA, transcript variant e-form (NM_033164.3) has been described ( 29 ) and f-form (NM_033163.3) was purchased from (OriGene Technologies, Rockville, MD).

Techniques: Functional Assay, Binding Assay, Over Expression, Injection, In Situ Hybridization, Staining, Control, Activity Assay, Western Blot, Construct, Transfection

Journal: Human Molecular Genetics

Article Title: Loss-of-function mutations in FGF8 can be independent risk factors for holoprosencephaly

doi: 10.1093/hmg/ddy106

Figure Lengend Snippet: The comorbid SHH p.S156R missense variant (Table 1, seen with the loss-of-function FGF8 p.T108M) is functionally normal in zebrafish. (A) Schematic drawing of SHH functional domains [SHH-N, signaling domain (red); SHH-C, autoprocessing domain (green)] and the location of tested variant positions. The previously described cholesterol addition site mutation p.C198S and p.T267I were selected as hypomorphic and severe loss-of-function positive controls, respectively (60). (B) Lateral and ventral view of a representative 10 pg of SHH mRNA-injected embryo at 28 hpf (right panels). The yellow arrow points to the appearance of a normal sized zebrafish eye (gfp injection control), while the red arrow indicates a small eye (microphthalmia, right panels). (C) Quantitative measurements of control (gfp), wt and variants SHH-injected embryos. Yellow represents embryos with a normal phenotype and gray is for small eye (microphthalmia) embryos. Error bars represent SEM, *** indicates P < 0.001 when compared with SHH wt based on Student t-test. Quantification data in this study are described in Supplementary Material, Table S2.

Article Snippet: Expression constructs The FGF8 cDNA, transcript variant e-form (NM_033164.3) has been described ( 29 ) and f-form (NM_033163.3) was purchased from (OriGene Technologies, Rockville, MD).

Techniques: Variant Assay, Functional Assay, Mutagenesis, Injection, Control

Journal: Journal of translational medicine

Article Title: Mutations in fibroblast growth factor (FGF8) and FGF10 identified in patients with conotruncal defects.

doi: 10.1186/s12967-020-02445-2

Figure Lengend Snippet: Fig. 1 Sequences of FGF8 and FGF10 mutants identified in patients with CTD and controls. a, c, e Chromatograms of normal controls. b, d Chromatograms of the two heterozygous variants. f Deletion mutation in FGF10. Arrows indicate the nucleotide changes and the deletion

Article Snippet: The human FGF8 (pCMV6-Entry-FGF8, Myc-DDKtagged) and FGF10 (pCMV6-Entry-FGF10, Myc-DDKtagged) cDNA plasmids were purchased from Origene (Rockville, MD, USA).

Techniques: Mutagenesis

Journal: Journal of translational medicine

Article Title: Mutations in fibroblast growth factor (FGF8) and FGF10 identified in patients with conotruncal defects.

doi: 10.1186/s12967-020-02445-2

Figure Lengend Snippet: Fig. 2 Distribution and conservation of mutations in FGF8 and FGF10. a, b Alignment of multiple FGF8 and FGF10 protein sequences among species. c, d Structure of the FGF8 and FGF10 proteins and the location of the genetic variations in this study

Article Snippet: The human FGF8 (pCMV6-Entry-FGF8, Myc-DDKtagged) and FGF10 (pCMV6-Entry-FGF10, Myc-DDKtagged) cDNA plasmids were purchased from Origene (Rockville, MD, USA).

Techniques:

Journal: Journal of translational medicine

Article Title: Mutations in fibroblast growth factor (FGF8) and FGF10 identified in patients with conotruncal defects.

doi: 10.1186/s12967-020-02445-2

Figure Lengend Snippet: Fig. 3 The expression and intracellular localization of wild-type and mutated FGF8. Relative mRNA expression of wild-type and variants of FGF8 in HCM (a) and HEK 293T (c), respectively (n = 3). GAPDH was used as an internal control. Western blot analysis and density quantitation in HCM (b) and HEK 293T (d) transfected with the blank vector, wild-type, and mutated FGF8. GAPDH and β-actin were used as an internal control in HCM and HEK293T, respectively (n = 3). e Immunofluorescence staining of wild-type and variants of FGF8. Images represented here were obtained from 3 biological replicates. Scale bar 20 μm

Article Snippet: The human FGF8 (pCMV6-Entry-FGF8, Myc-DDKtagged) and FGF10 (pCMV6-Entry-FGF10, Myc-DDKtagged) cDNA plasmids were purchased from Origene (Rockville, MD, USA).

Techniques: Expressing, Control, Western Blot, Quantitation Assay, Transfection, Plasmid Preparation, Immunofluorescence, Staining

Journal: Journal of translational medicine

Article Title: Mutations in fibroblast growth factor (FGF8) and FGF10 identified in patients with conotruncal defects.

doi: 10.1186/s12967-020-02445-2

Figure Lengend Snippet: Fig. 5 Functional analysis of wild-type and mutated FGF8. a Relative amount of FGF8 in supernatants. b Effects of mutant and wild-type FGF8 on cell viability. c, d The variant effect of FGF8 on PEA3 expression (n = 3), GAPDH was used as an internal control

Article Snippet: The human FGF8 (pCMV6-Entry-FGF8, Myc-DDKtagged) and FGF10 (pCMV6-Entry-FGF10, Myc-DDKtagged) cDNA plasmids were purchased from Origene (Rockville, MD, USA).

Techniques: Functional Assay, Mutagenesis, Variant Assay, Expressing, Control

Journal: Journal of translational medicine

Article Title: Mutations in fibroblast growth factor (FGF8) and FGF10 identified in patients with conotruncal defects.

doi: 10.1186/s12967-020-02445-2

Figure Lengend Snippet: Fig. 8 Scheme of the regulatory mechanisms involving FGF8 and FGF10 in the pathogenesis of CTDs. The upward arrow indicates increased expression, the downward arrow indicates decreased secretion, and the minus sign indicates inhibition

Article Snippet: The human FGF8 (pCMV6-Entry-FGF8, Myc-DDKtagged) and FGF10 (pCMV6-Entry-FGF10, Myc-DDKtagged) cDNA plasmids were purchased from Origene (Rockville, MD, USA).

Techniques: Expressing, Inhibition

Journal: Journal of translational medicine

Article Title: Mutations in fibroblast growth factor (FGF8) and FGF10 identified in patients with conotruncal defects.

doi: 10.1186/s12967-020-02445-2

Figure Lengend Snippet: Fig. 7 Dynamic expression of FGF8 and FGF10 in human embryonic stem cells and human embryonic cardiac tissue. a, b Relative expression of FGF8 and FGF10 during the differentiation of human embryonic stem cells to cardiomyocytes. c Expression level of FGF8 and FGF10 in the human embryonic heart, d, e Immunohistochemistry of FGF8 and FGF10 in human embryos at Carnegie stge13

Article Snippet: The human FGF8 (pCMV6-Entry-FGF8, Myc-DDKtagged) and FGF10 (pCMV6-Entry-FGF10, Myc-DDKtagged) cDNA plasmids were purchased from Origene (Rockville, MD, USA).

Techniques: Expressing, Immunohistochemistry

Journal: Oncogene

Article Title: Retinoid-suppressed phosphorylation of RARalpha mediates the differentiation pathway of osteosarcoma cells.

doi: 10.1038/onc.2010.50

Figure Lengend Snippet: Figure 7 RARa hypophosphorylation induces FGF8f expression. (a) Primers designed for analysis of total FGF8 and FGF8 isoforms (the diagram does not reflect the actual dimension flanked by the primers). (b, c) RT–PCR analysis of FGF8 isoforms and FGF8f expression. (d, e) WB analysis of FGF8f proteins in the lysates (panel d) and in the medium of U2OS cells expressing RARaS77A (panel e).

Article Snippet: Lentiviral plasmid construction, virion production and transduction The RARaS77A cDNA (Luo et al., 2007) and FGF8f cDNA (OriGene, Rockville, MD, USA) were cloned into pCCL-cMNDU3c-X2-PKG-EGFP lentiviral vector (Luo et al., 2007).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Oncogene

Article Title: Retinoid-suppressed phosphorylation of RARalpha mediates the differentiation pathway of osteosarcoma cells.

doi: 10.1038/onc.2010.50

Figure Lengend Snippet: Figure 8 FGF8f is a downstream target of RARa hypophosphorylation in RA-induced osteoblastic differentiation. (a) Morphology of U2OS cells transduced with FGF8f. (b) Proliferation analysis by cell count. (c, d) RT–PCR depicted expression of differentiation regulators. (e, f) WB analyses of FGF8f and OPN proteins. (g) FGF8-siRNA decreased FGF8f mRNA levels. B-peptide, blocking peptide; C-siRNA, control siRNA. (h) FGF8-siRNA decreased levels of OPN mRNA. (i) FGF8-siRNA counteracted the effects of either RA or FGF8f on inhibition of proliferation. siRNA vs vehicle: *P ¼ 0.000; **Po0.005. (j) FGF8 antibodies neutralized the inhibition effect of FGF8f on cell proliferation. FGF8 antibodies vs PBS: #Po0.02; *Po0.04; **Po0.003.

Article Snippet: Lentiviral plasmid construction, virion production and transduction The RARaS77A cDNA (Luo et al., 2007) and FGF8f cDNA (OriGene, Rockville, MD, USA) were cloned into pCCL-cMNDU3c-X2-PKG-EGFP lentiviral vector (Luo et al., 2007).

Techniques: Transduction, Cell Counting, Reverse Transcription Polymerase Chain Reaction, Expressing, Blocking Assay, Control, Inhibition

Journal: Oncogene

Article Title: Retinoid-suppressed phosphorylation of RARalpha mediates the differentiation pathway of osteosarcoma cells.

doi: 10.1038/onc.2010.50

Figure Lengend Snippet: Figure 9 Hypothetical network of differentiation response path- ways coordinated by RA-RARa-FGF8f signal transduction during osteoblastic differentiation.

Article Snippet: Lentiviral plasmid construction, virion production and transduction The RARaS77A cDNA (Luo et al., 2007) and FGF8f cDNA (OriGene, Rockville, MD, USA) were cloned into pCCL-cMNDU3c-X2-PKG-EGFP lentiviral vector (Luo et al., 2007).

Techniques: Transduction