Journal: The FASEB Journal

Article Title: Loss of the RNA Binding Protein HuR in Early Murine Limb Mesenchyme Does Not Affect Development but Leads to Impaired Bone Homeostasis in Adulthood

doi: 10.1096/fj.202500780RR

Figure Lengend Snippet: Effect of germline Cre recombination in HuR fl/fl mice (Elavl1 KO) through maternal inheritance of Prx1‐Cre. (A) Whole‐mount embryos at E13.5 stained with alcian blue. (B) Toluidine blue‐stained sections of E13.5 embryo forelimbs and hindlimbs. (C) Immunofluorescence detection of HuR in limb sections from E13.5 embryos. (D) Brightfield micrographs demonstrating morphology changes in Control and Elavl1 KO embryos isolated from E9.5 to E11.5. Scale bar = 500 μm. (E) Whole‐mount in situ hybridization chain reaction detection of limb bud markers mRNA in E10.5 and E11.5 embryos. Asterisk: Limb bud, Triangle: Apical ectodermal ridge, Arrowhead: The zone of polarizing activity. Scale bar = 150 μm. (F) Whole‐mount immunofluorescence detection of Fgf8 protein in E10.5 embryos. Asterisk: Limb bud, Triangle: Apical ectodermal ridge. Scale bar = 150 μm.

Article Snippet: Embryos were fixed in 4% formaldehyde for 1 h at 4°C, washed, and stored in 0.0025% Triton X‐100 in PBS (PBS‐Tr) at 4° C. Prior to immunofluorescence staining, embryos were permeabilized with 0.25% Triton X‐100 in PBS for 30 min and then blocked with 2% bovine serum albumin in PBS solution for 1 h before incubation with 1:100 Fgf8 antibody (MAB323, R&D Systems, UK) overnight at 4°C.

Techniques: Staining, Immunofluorescence, Control, Isolation, In Situ Hybridization, Activity Assay

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