Journal: Cancer Research

Article Title: Signal Transducers and Activators of Transcription-3 Binding to the Fibroblast Growth Factor Receptor Is Activated by Receptor Amplification

doi: 10.1158/0008-5472.can-09-3033

Figure Lengend Snippet: Figure 2. Serine STAT3 activation is a downstream effect of FGFR signaling. MCF7 cells were treated with inhibitors or DMSO for 30 min and stimulated with FGF1 and heparin. Whole cell lysates were analyzed by Western blotting. Densitometric analysis of Western blots for pSerSTAT3 normalized to STAT3 was performed from three separate experiments. Error bars, SD. Statistical significance was determined by t test comparison between each sample treated with inhibitor and sample stimulated with FGF1 (*, P < 0.05).

Article Snippet: Rabbit anti-Bek, rabbit anti-Flg, rabbit anti-ERK1, mouse anti-phosphorylated ERK1, rabbit anti-JunB, and rabbit c-fos antibodies were obtained from Santa Cruz Biotechnology, Inc. Rabbit anti-phosphorylated Jak2 (Tyr1007/1008), rabbit anti-Jak2, mouse anti-Src, rabbit anti-phosphorylated Src family (Tyr416), mouse anti-STAT3, rabbit anti-phosphorylated STAT3 (Tyr705), rabbit antiphosphorylated STAT3 (Ser727), rabbit anti-phosphorylated STAT5 (Tyr694), mouse anti-phosphorylated FGFR (Tyr653/654), rabbit anti–c-myc, and mouse myc-tag antibodies were obtained from Cell Signaling Technology.

Techniques: Activation Assay, Western Blot, Comparison

Journal: Cancer Research

Article Title: Signal Transducers and Activators of Transcription-3 Binding to the Fibroblast Growth Factor Receptor Is Activated by Receptor Amplification

doi: 10.1158/0008-5472.can-09-3033

Figure Lengend Snippet: Figure 3. Tyrosine STAT3 phosphorylation depends on high levels of FGFR. A, MCF7 and SUM-52PE cells were serum-starved and stimulated with FGF1 and heparin. Whole cell lysates were analyzed by Western blotting. Densitometric analysis of Western blots for FGFR2 normalized to α-tubulin was performed from three separate experiments. Error bars, SD. Statistical significance between samples was determined by t test. B, SUM-52PE cells were serum-starved and stimulated with FGF7 and heparin as indicated. Whole cell lysates were analyzed by Western blotting. Densitometric analysis of Western blots for pTyr- and pSerSTAT3 normalized to STAT3 was performed from one experiment. C, SUM-52PE cells were serum-starved, treated with inhibitors as indicated or DMSO for 30 min, and stimulated with FGF7 and heparin. Whole cell lysates were analyzed by Western blotting. D, SUM-52PE cells were transfected with FGFR2 siRNA oligonucleotides. Cells were serum-starved, stimulated with FGF7 and heparin. Whole cell lysates were analyzed by Western blotting. Densitometric analysis of Western blots for p653/654FGFR normalized to α-tubulin and pTyrSTAT3 normalized to STAT3 was performed from three separate experiments. Error bars, SD.

Article Snippet: Rabbit anti-Bek, rabbit anti-Flg, rabbit anti-ERK1, mouse anti-phosphorylated ERK1, rabbit anti-JunB, and rabbit c-fos antibodies were obtained from Santa Cruz Biotechnology, Inc. Rabbit anti-phosphorylated Jak2 (Tyr1007/1008), rabbit anti-Jak2, mouse anti-Src, rabbit anti-phosphorylated Src family (Tyr416), mouse anti-STAT3, rabbit anti-phosphorylated STAT3 (Tyr705), rabbit antiphosphorylated STAT3 (Ser727), rabbit anti-phosphorylated STAT5 (Tyr694), mouse anti-phosphorylated FGFR (Tyr653/654), rabbit anti–c-myc, and mouse myc-tag antibodies were obtained from Cell Signaling Technology.

Techniques: Phospho-proteomics, Western Blot, Transfection

Journal: Molecules

Article Title: Comparison of the Interactions of Different Growth Factors and Glycosaminoglycans

doi: 10.3390/molecules24183360

Figure Lengend Snippet: Figure 3. Bar graphs of normalized different growth factors binding preference to surface heparin by competing with different size of heparin oligosaccharides (from fp4 to dp18). (A) FGF2, (B) FGF7, (C) FGF10, (D) HGF, (E) TGFβ-1. Concentrations were 200, 100, 100, 20, and 50 nM for FGF2, FGF7, FGF10, HGF and TGFβ-1, respectively, and concentrations of heparin oligosaccharides were 1000 nM. All bar graphs based on triplicate experiments.

Article Snippet: Recombinant human FGF2 was a gift from Amegen; FGF7 and FGF10 were generously provided by Professor Mohammadi from NYU; HGF and TGF β-1 were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Binding Assay

Journal: Molecules

Article Title: Comparison of the Interactions of Different Growth Factors and Glycosaminoglycans

doi: 10.3390/molecules24183360

Figure Lengend Snippet: Figure 4. Bar graphs of normalized different growth factors binding preference to surface heparin by competing with different chemical modified heparin in solution. (A) FGF2, (B) FGF7, (C) FGF10, (D) HGF, (E) TGFβ-1. Concentrations were 200, 100, 100, 20, and 50 nM for FGF2, FGF7, FGF10, HGF and TGFβ-1, respectively, and concentrations of different modified heparin were 1000 nM. All bar graphs based on triplicate experiments.

Article Snippet: Recombinant human FGF2 was a gift from Amegen; FGF7 and FGF10 were generously provided by Professor Mohammadi from NYU; HGF and TGF β-1 were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Binding Assay

Journal: Molecules

Article Title: Comparison of the Interactions of Different Growth Factors and Glycosaminoglycans

doi: 10.3390/molecules24183360

Figure Lengend Snippet: Figure 5. Bar graphs of normalized different growth factors binding preference to surface heparin by competing with different GAGs in solution. (A) FGF2, (B) FGF7, (C) FGF10, (D) HGF, (E) TGFβ-1. Concentrations were 200, 100, 100, 20, and 50 nM for FGF2, FGF7, FGF10, HGF and TGFβ-1, respectively and concentrations of GAGs were 1000 nM. All bar graphs based on triplicate experiments.

Article Snippet: Recombinant human FGF2 was a gift from Amegen; FGF7 and FGF10 were generously provided by Professor Mohammadi from NYU; HGF and TGF β-1 were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Binding Assay

Journal: Reproduction

Article Title: Culture conditions antagonize lineage-promoting signaling in the mouse blastocyst

doi: 10.1530/rep-20-0107

Figure Lengend Snippet: Figure 1 BSA antagonizes signaling by exogenous FGF4. (A) Experimental design for embryos shown and analyzed in this figure. Red = NANOG indicative of epiblast cell fate, green = SOX17 indicative of primitive endoderm cell fate. (B) Immunostaining for SOX17 to identify primitive endoderm (PE) cells and NANOG to identify epiblast (EPI) cells in embryos cultured for 66 hours starting from two-cell stage in KSOM with 1 mg/mL BSA or without BSA (Millipore-Sigma MR-121-D and MR-107-D, respectively), but with 1 mg/mL added PVA (Sigma 360627). n = number of embryos examined. P = Student’s t-test. n.s. = not significant (P > 0.2). (C) Quantification of the total inner cell mass (ICM) and trophectoderm (TE) cells in embryos cultured in indicated conditions and proportion of ICM cells contributing to PE and EPI in these embryos. Each symbol represents a single embryo, column = mean, and error bars = standard deviations. P = Student’s t-test. n.s. = not significant (P > 0.2). (D) Immunostaining for SOX17 and NANOG in embryos cultured in KSOM + 150 ng/mL FGF4 (R&D Systems 235-F4-025) and 1 µg/mL heparin (Sigma H3149) in the presence or absence of 1 mg/mL BSA. Each symbol represents a single embryo, column = mean, and error bars = s.d. P = Student’s t-test. n.s. = not significant (P > 0.2). (E) Quantification of lineage specification in embryos cultured in conditions indicated. Each symbol represents a single embryo, column = mean, and error bars = s.d. (F) Response of embryos cultured in KSOM without 1 mg/mL BSA to low dose of exogenous FGF4 and 1 µg/mL heparin. Each symbol represents a single embryo, column = mean, and error bars = s.d. P = Student’s t-test.

Article Snippet: = not significant (P > 0.2). (D) Immunostaining for SOX17 and NANOG in embryos cultured in KSOM + 150 ng/mL FGF4 (R&D Systems 235-F4-025) and 1 μg/mL heparin (Sigma H3149) in the presence or absence of 1 mg/mL BSA.

Techniques: Immunostaining, Cell Culture

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: PLoS ONE

Article Title: Fibroblast-growth factor 23 promotes terminal differentiation of ATDC5 cells

doi: 10.1371/journal.pone.0174969

Figure Lengend Snippet: Primer sequences for RT-qPCR.

Article Snippet: When required, after serum deprivation, cells were stimulated by 100 ng/mL of recombinant mouse FGF23 (rFGF23, R&D Systems, UK) for 24h at 7, 14, 21 or 28 days of culture and treated with 0.5 or 1 μM of PD173074 (Sigma-Aldrich, France), a pan-FGFR inhibitor, during the culture.

Techniques:

Journal: PLoS ONE

Article Title: Fibroblast-growth factor 23 promotes terminal differentiation of ATDC5 cells

doi: 10.1371/journal.pone.0174969

Figure Lengend Snippet: Total RNA was extracted from ATDC5 cultured in ITS conditions for 0, 7, 14, 21 and 28 days, then reverse transcribed into cDNA and analysed by real-time PCR. The relative abundance of FGF23 (A) was normalized to RPS29 mRNA. Comparison was made by using the ΔΔCt method with the fold value of reference (fold = 1) assigned to D0. *: p < 0.05 vs D0, **: p< 0,01 vs D0. Data are expressed as mean ± SD, n = 4. FGF23 production in media was quantified by ELISA (B), **: p< 0,01 vs D0, ***: p< 0,001, ND: Non Detectable. Total proteins were extracted from ATDC5 (0, 7, 14, 21 or 28 days post-insulin) and 10 μg was subjected to SDS-PAGE with FGF23 (1/200) antibody; ß-actin was used as loading control (C).

Article Snippet: When required, after serum deprivation, cells were stimulated by 100 ng/mL of recombinant mouse FGF23 (rFGF23, R&D Systems, UK) for 24h at 7, 14, 21 or 28 days of culture and treated with 0.5 or 1 μM of PD173074 (Sigma-Aldrich, France), a pan-FGFR inhibitor, during the culture.

Techniques: Cell Culture, Reverse Transcription, Real-time Polymerase Chain Reaction, Comparison, Enzyme-linked Immunosorbent Assay, SDS Page, Control

Journal: PLoS ONE

Article Title: Fibroblast-growth factor 23 promotes terminal differentiation of ATDC5 cells

doi: 10.1371/journal.pone.0174969

Figure Lengend Snippet: Total RNA was extracted from ATDC5 cultured in ITS conditions for 14 or 28 days and stimulated or not with 100ng/mL mouse rFGF23 for 24h, then reverse transcribed into cDNA and analysed by real-time PCR. The relative abundance at D14 and D28 of FGF23 (A,B), COLX (C,D) and MMP13 (E,F) was normalized to RPS29 mRNA. Comparison was made by using the ΔΔCt method with the fold value of reference (fold = 1) assigned to D0. *: p < 0.05 vs D0, # p<0.05 vs ITS. Data are expressed as mean ± SD, n = 3.

Article Snippet: When required, after serum deprivation, cells were stimulated by 100 ng/mL of recombinant mouse FGF23 (rFGF23, R&D Systems, UK) for 24h at 7, 14, 21 or 28 days of culture and treated with 0.5 or 1 μM of PD173074 (Sigma-Aldrich, France), a pan-FGFR inhibitor, during the culture.

Techniques: Cell Culture, Reverse Transcription, Real-time Polymerase Chain Reaction, Comparison

Journal: PLoS ONE

Article Title: Fibroblast-growth factor 23 promotes terminal differentiation of ATDC5 cells

doi: 10.1371/journal.pone.0174969

Figure Lengend Snippet: ATDC5 were fixed with 4% PFA, and then stained by Alizarin red for 45min after 21 or 28 days of insulin stimulation with or without 100 ng/mL of mouse rFGF23. Images presented are representative of 5 independent experiments (A). ATDC5 micromasses were cultured for 14 days with insulin stimulation in the presence of 3 mM Pi or 100 ng/mL rFGF23 or both. Then they were fixed with 4% PFA and stained by Alizarin red for 45min. Images presented are representative of 3 independent experiments (B). Total RNA was extracted from ATDC5 cultured in ITS conditions for 14 days, then reverse transcribed into cDNA and analysed by real-time PCR and compared to ITS. The relative abundance of COL X, MMP13 and FGF23 were normalized to RPS29 mRNA (C, D, E). Comparison was made by using the ΔΔCt method with the fold value of reference (fold = 1) assigned to ITS. *: p < 0.05 vs ITS, #: p < 0.05 vs ITS + Pi and £: p < 0.05 vs ITS + rFGF23. Data are expressed as mean ± SD, n = 3.

Article Snippet: When required, after serum deprivation, cells were stimulated by 100 ng/mL of recombinant mouse FGF23 (rFGF23, R&D Systems, UK) for 24h at 7, 14, 21 or 28 days of culture and treated with 0.5 or 1 μM of PD173074 (Sigma-Aldrich, France), a pan-FGFR inhibitor, during the culture.

Techniques: Staining, Cell Culture, Reverse Transcription, Real-time Polymerase Chain Reaction, Comparison

Journal: PLoS ONE

Article Title: Fibroblast-growth factor 23 promotes terminal differentiation of ATDC5 cells

doi: 10.1371/journal.pone.0174969

Figure Lengend Snippet: Effect of specific inhibition of FGFRs activation during ATDC5 differentiation process (Day 14).

Article Snippet: When required, after serum deprivation, cells were stimulated by 100 ng/mL of recombinant mouse FGF23 (rFGF23, R&D Systems, UK) for 24h at 7, 14, 21 or 28 days of culture and treated with 0.5 or 1 μM of PD173074 (Sigma-Aldrich, France), a pan-FGFR inhibitor, during the culture.

Techniques: Inhibition, Activation Assay, Expressing

Journal: PLoS ONE

Article Title: Fibroblast-growth factor 23 promotes terminal differentiation of ATDC5 cells

doi: 10.1371/journal.pone.0174969

Figure Lengend Snippet: Effect of specific inhibition of FGFRs activation during ATDC5 differentiation process (Day 28).

Article Snippet: When required, after serum deprivation, cells were stimulated by 100 ng/mL of recombinant mouse FGF23 (rFGF23, R&D Systems, UK) for 24h at 7, 14, 21 or 28 days of culture and treated with 0.5 or 1 μM of PD173074 (Sigma-Aldrich, France), a pan-FGFR inhibitor, during the culture.

Techniques: Inhibition, Activation Assay, Expressing

Journal: PLoS ONE

Article Title: Fibroblast-growth factor 23 promotes terminal differentiation of ATDC5 cells

doi: 10.1371/journal.pone.0174969

Figure Lengend Snippet: Total RNA was extracted from ATDC5 cultured in ITS conditions for 28 days pre-treated or not with 1 μM PD173074 one hour before stimulation with 100 ng/ml of rFGF23 (24h for RNA or 48h for proteins), then reverse transcribed into cDNA and analysed by real-time PCR. The relative abundance of FGF23 (A), MMP13 (B) and COL X (C) was normalized to RPS29 mRNA. Comparison was made by using the ΔΔCt method with the fold value of reference (fold = 1) assigned to D0. *: p < 0.05 vs D0, # p< 0.05 vs ITS, £: p< 0.01 vs rFGF23. Data are expressed as mean ± SD, n = 3.

Article Snippet: When required, after serum deprivation, cells were stimulated by 100 ng/mL of recombinant mouse FGF23 (rFGF23, R&D Systems, UK) for 24h at 7, 14, 21 or 28 days of culture and treated with 0.5 or 1 μM of PD173074 (Sigma-Aldrich, France), a pan-FGFR inhibitor, during the culture.

Techniques: Cell Culture, Reverse Transcription, Real-time Polymerase Chain Reaction, Comparison