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: Nature genetics

Article Title: FGF9 monomer/dimer equilibrium regulates extracellular matrix affinity and tissue diffusion

doi: 10.1038/ng.316

Figure Lengend Snippet: Missense mutation in the Fgf9 gene of Eks mice. ( a ) Nucleotide sequence of the Fgf9 cDNA derived from +/+ and Eks/Eks mice. Ek s mutants have an A to C substitution at position 428, which results in the replacement of Asn143 with Thr. The Eks missense mutation is indicated by the asterisk and the corresponding amino acid is shown in purple. ( b ) Structure-based sequence alignment of human FGFs. The amino acid sequence surrounding the N143T mutation in FGF9 Eks and that of its corresponding domain in other human FGF family proteins are aligned based on sequence identity. The Asn143 residue in FGF9 is highly conserved among most FGF proteins (purple box). The asterisk denotes the site of Eks mutation.

Article Snippet: 5,000 cells were plated per well in a 96-well assay plate in media containing varying concentrations of FGF9 and heparin (Wako).

Techniques: Mutagenesis, Sequencing, Derivative Assay

Journal: Nature genetics

Article Title: FGF9 monomer/dimer equilibrium regulates extracellular matrix affinity and tissue diffusion

doi: 10.1038/ng.316

Figure Lengend Snippet: Fgf9 Eks/Eks mice phenocopy Fgfr s gain-of-function mutants. ( a–l ) Defects in early specification of prospective elbow joints in Fgf9 Eks/Eks embryos. Hematoxylin and eosin staining ( a, b, g, h ) and in situ detection of Gdf5 ( c, d, i, j ) and Col2a1 ( e, f, k, l ) in the forelimb buds of Fgf9 +/+ and Fgf9 Eks/Eks embryos at E10.5 and E11.5. In Fgf9 +/+ embryos there was Gdf5 expression at the prospective elbow joint ( i ), which was demarcated as the gap of Col2a1 expression ( k , arrow), at E11.5. In Fgf9 Eks/Eks embryos there was not Gdf5 expression at the prospective elbow joint ( j ). Scale bars, 100 µm. ( m–b’ ) Ectopic osteogenesis at the coronal sutures in Fgf9 Eks/Eks fetuses. Hematoxylin and eosin staining ( m, n, u, v ) and von Kossa staining ( o, p, w, x ) and in situ detection of Spp1 ( q, r, y, z ) and Runx2 ( s, t, a’, b’ ) in the coronal suture of Fgf9 +/+ and Fgf9 Eks/Eks fetuses at E15.5 and E16.5. Note the ectopic ossification in the suture of Fgf9 Eks/Eks at E16.5 ( v, x, z, b’ ). fb, frontal bone; pb, parietal bone. Scale bars, 100 µm.

Article Snippet: 5,000 cells were plated per well in a 96-well assay plate in media containing varying concentrations of FGF9 and heparin (Wako).

Techniques: Staining, In Situ, Expressing

Journal: Nature genetics

Article Title: FGF9 monomer/dimer equilibrium regulates extracellular matrix affinity and tissue diffusion

doi: 10.1038/ng.316

Figure Lengend Snippet: The Eks mutation affects dimerization of FGF9. ( a, b ) Sedimentation equilibrium analysis of FGF9 WT and FGF9 Eks . Ln A 280nm versus radius 2 during sedimentation equilibrium at 16,000 rpm at 20°C is indicated using 0.4 mg/ml of FGF9 WT ( a ) and FGF9 Eks ( b ). The residuals are shown in the upper panels. ( c, d ) Sedimentation velocity analysis of FGF9 WT and FGF9 Eks . The sedimentation coefficient distribution for FGF9 WT ( c ) and FGF9 Eks ( d ) at the concentrations of 0.2, 0.3 and 0.4 mg/ml are shown. ( e ) Analytical gel filtration chromatography of FGF9 WT and FGF9 Eks . FGF9 WT or FGF9 Eks applied separately to Superdex 75 10/300 GL columns. Eluted FGF9 WT and FGF9 Eks were identified by absorbance at 280 nm. Arrows indicate the position of the size standards: 67 kDa, albumin; 43 kDa, ovalbumin; 25 kDa, chymotrypsinogen; 13.7 kDa, ribonuclease A.

Article Snippet: 5,000 cells were plated per well in a 96-well assay plate in media containing varying concentrations of FGF9 and heparin (Wako).

Techniques: Mutagenesis, Sedimentation, Filtration, Chromatography

Journal: Nature genetics

Article Title: FGF9 monomer/dimer equilibrium regulates extracellular matrix affinity and tissue diffusion

doi: 10.1038/ng.316

Figure Lengend Snippet: The Eks mutation affects the mitogenic activity of FGF9. ( a–g ) Dose dependent changes in mitogenic activity of FGF9 WT and FGF9 Eks . BaF3 cells expressing exogenous FGFR1b, 1c, 2b, 2c, 3b, 3c or 4 were treated with increasing concentrations of FGF9 WT or FGF9 Eks in the presence of 1 µg/ml heparin. Cell proliferation was determined by [ 3 H]thymidine incorporation after 36 hours in culture. ( h–n ) Heparin-dependent changes in mitogenic activity of FGF9 WT and FGF9 Eks . BaF3 cells expressing the respective FGFR were treated with increasing concentrations of heparin in the presence of 0.2 nM FGF9 WT or FGF9 Eks . Cell proliferation was determined as above. Data are represented as mean ± s.e.m. of triplicate assays. These results are representative of at least two independent experiments.

Article Snippet: 5,000 cells were plated per well in a 96-well assay plate in media containing varying concentrations of FGF9 and heparin (Wako).

Techniques: Mutagenesis, Activity Assay, Expressing

Journal: Nature genetics

Article Title: FGF9 monomer/dimer equilibrium regulates extracellular matrix affinity and tissue diffusion

doi: 10.1038/ng.316

Figure Lengend Snippet: The Eks mutation reduces FGF9 affinity for heparin by impairing its dimerization. ( a ) Chromatographic analysis of the affinities of FGF9 WT and FGF9 Eks for heparin. FGF9 WT or FGF9 Eks were loaded onto a heparin-conjugated agarose column and eluted with a linear gradient of NaCl from 120 mM to 2.0 M (black line). Elution profiles of FGF9 WT and FGF9 Eks were determined by monitoring absorbance at 280 nm. ( b, c ) Surface plasmon resonance analysis of the affinities of FGF9 WT and FGF9 Eks for heparin. Sensorgrams indicating the interaction of FGF9 WT ( b ) and FGF9 Eks ( c ) with immobilized heparin were determined at different concentrations. The biosensor chip response is indicated on the y-axis (RU) as a function of time (x-axis) at room temperature. ( d–g ) The most probable solution structures of dimeric FGF9 WT -heparin ( d ), dimeric FGF9 Eks -heparin ( e ), monomeric FGF9 WT -heparin ( f ) and monomeric FGF9 Eks -heparin ( g ) complexes deduced by MD simulations. Heparin and protein residues that form important hydrogen bonds are drawn in ball and stick and space-filling modes. The single-letter amino acid code, residue number and chain code are indicated for each of these residues. Computed binding free energy of each complex is shown under the respective illustrated structure. Data are represented as mean ± s.d. of energies obtained from 200 MD snapshots in respective MD trajectory.

Article Snippet: 5,000 cells were plated per well in a 96-well assay plate in media containing varying concentrations of FGF9 and heparin (Wako).

Techniques: Mutagenesis, SPR Assay, Binding Assay

Journal: Nature genetics

Article Title: FGF9 monomer/dimer equilibrium regulates extracellular matrix affinity and tissue diffusion

doi: 10.1038/ng.316

Figure Lengend Snippet: FGF9 Eks can inhibit joint and suture development as well as FGF9 WT . ( a–g ) Inhibition of knee joint development induced by ectopic expression of Fgf9 Eks as well as Fgf9 WT . Hindlimb buds of HH stage 17 chickens were infected with RCAS- Fgf9 WT , RCAS- Fgf9 Eks , or empty RCAS virus. ( a, b ) Fgf9 expression was examined by in situ hybridization 2 days after infection. ( c–g ) Respective knee joints (arrows) were examined by Alcian blue staining ( c, d, f ) and hematoxylin and eosin staining of sections through the knee joint ( e, g ) 5 days after infection. f, femur; t, tibia. ( h–k ) Inhibition of coronal suture development by the ectopic presence of FGF9 Eks well as FGF9 WT . FGF9 WT or FGF9 Eks beads were implanted onto the coronal suture at E15 mice and the Spp1 expression was examined by in situ hybridization 24 hours after implantation. On the operated sides with FGF9 WT ( h ) and FGF9 Eks ( j ) bead implants, there was overlap of Spp1 expression in the frontal and parietal bones, which was not seen on the unoperated sides ( i, k ). fb, frontal bone; pb, parietal bone.

Article Snippet: 5,000 cells were plated per well in a 96-well assay plate in media containing varying concentrations of FGF9 and heparin (Wako).

Techniques: Inhibition, Expressing, Infection, In Situ Hybridization, Staining

Journal: Nature genetics

Article Title: FGF9 monomer/dimer equilibrium regulates extracellular matrix affinity and tissue diffusion

doi: 10.1038/ng.316

Figure Lengend Snippet: Ectopic FGF9 Eks signaling due to its hyper diffusibility. ( a–e ) Increased diffusibility of FGF9 Eks in the skull bone anlagen. FGF9 WT or FGF9 Eks beads were implanted onto the coronal suture at E15 mice and Spp1 expression was examined by whole-mount in situ hybridization 24 hours after implantation. On the operated sides with FGF9 WT ( a ) and FGF9 Eks ( c ) bead implants, we observed well-defined intense signals in the frontal and parietal bone anlagen around the implants, which were not seen on the unoperated side ( b, d ). This domain with intense Spp1 signals reflects diffusibility of exogenous FGF9 proteins. We therefore compared diffusibility of FGF9 WT and FGF9 Eks based on the induced expression domain of Spp1 ( e ). The diffusion areas (%) in the frontal and parietal bone anlagen area were estimated from the area ratio of the intense Spp1 expression against the frontal and parietal bone anlagen. Data are represented as mean ± s.e.m. of six operations. FGF9 Eks is more diffusible than FGF9 WT . Significance was determined by two-tailed Student’s t -test. ( f–h ) Increased diffusibility of FGF9 Eks in the forelimb bud. FGF9 WT or FGF9 Eks beads were implanted into forelimb buds of Fgf9 −/− embryos of E10.5 mice. Diffusion of exogenous FGF9 WT ( f ) and FGF9 Eks ( g ) after 2 hours was immunodetected using a FGF9 antibody. ( h ) The diffusion area of FGF9 Eks and FGF9 WT was measured at the level of the equator of the beads. Data are represented as mean (FGF9 WT =100%) ± s.e.m. of four (FGF9 WT ) or five (FGF9 Eks ) operations. FGF9 Eks is also more diffusible than FGF9 WT in limb buds. Significance was determined by one-tailed Student’s t -test. ( i–x ) The downstream target genes of FGF signaling, Etv4 and Etv5 , are expressed ectopically in the prospective elbow joint in Fgf9 Eks/Eks mice. Counterstaining with Giemsa ( i, j, q, r ) and in situ detection of Col2a1 ( k, l, s, t ), Etv4 ( m, n, u, v ) and Etv5 ( o, p, w, x ) in the forelimb buds of Fgf9 +/+ and Fgf9 Eks/Eks embryos at E10.5 and E11.5. There was ectopic expression of Etv4 ( n, v ) and Etv5 ( p, x ), in the cartilaginous condensation including the prospective elbow joint position, which was demarcated as the gap of Col2a1 expression ( s , arrow), in Fgf9 Eks/Eks , which were not seen in Fgf9 +/+ mice ( m, o, u, w ). Scale bars, 100 µm. ( y ) A model for the pathogenic mechanism underlying elbow joint synosotsis in Fgf9 Eks/Eks mice. In Fgf9 Eks/Eks mice, ectopic FGF9 signaling due to hyper-diffusion of FGF9 Eks at the prospective elbow joint may inhibit the initiation of joint development. ( z ) A model for the pathogenic mechanism underlying premature fusion of the coronal suture in Fgf9 Eks/Eks mice. In Fgf9 Eks/Eks mice, ectopic FGF9 signaling due to hyper-diffusion of FGF9 Eks at the coronal suture may promote ectopic osteogenesis and subsequently induce premature fusion of the suture.

Article Snippet: 5,000 cells were plated per well in a 96-well assay plate in media containing varying concentrations of FGF9 and heparin (Wako).

Techniques: Expressing, In Situ Hybridization, Diffusion-based Assay, Two Tailed Test, One-tailed Test, In Situ

Journal: Nature genetics

Article Title: FGF9 monomer/dimer equilibrium regulates extracellular matrix affinity and tissue diffusion

doi: 10.1038/ng.316

Figure Lengend Snippet: FGF9 WT modulates FGF9 Eks action by forming FGF9 WT /FGF9 Eks heterodimers. ( a–c ) Proposed solution structures for FGF9 WT homodimer ( a ), FGF9 WT/ Eks heterodimer ( b ) and FGF9 Eks homodimer ( c ). Amino acid residues contributing to hydrogen bond formation involved in dimerization are drawn in ball and stick and space-filling modes. The single-letter amino acid code, residue number and chain code are indicated for each of these residues. Computed binding free energy of each dimer is shown under the respective illustrated structure. Data are represented as mean ± s.d. of energies obtained from 200 MD snapshots in respective MD trajectory. ( d ) FGF9 WT is capable of forming dimers with FGF9 Eks . The expression of FGF9 WT homodimers, FGF9 WT/ Eks heterodimers and FGF9 Eks homodimers was analyzed using IP/Western blots. The expression vectors for FLAG- or Myc-tagged FGF9 WT and FGF9 Eks were transfected into COS7 cells and culture supernatants were subjected to IP/Western analysis. ( e–h ) Less severe elbow joint synostosis in Fgf9 Eks/+ than Fgf9 Eks/− . Forelimbs from Fgf9 +/− , Fgf9 Eks/+ , Fgf9 Eks/− and Fgf9 Eks/Eks embryos at E17.5 were stained with Alcian blue and Alizarin red. Synostotic change is restricted to the cartilaginous component in Fgf9 Eks/+ embryos, whereas it is extended to the bony component in Fgf9 Eks/− , and Fgf9 Eks/Eks embryos. h, humerus; r, radius; u, ulna.

Article Snippet: 5,000 cells were plated per well in a 96-well assay plate in media containing varying concentrations of FGF9 and heparin (Wako).

Techniques: Binding Assay, Expressing, Western Blot, Transfection, Staining

Journal:

Article Title: Wnt and FGF signals interact to coordinate growth with cell fate specification during limb development

doi: 10.1242/dev.023176

Figure Lengend Snippet: (A–G) Alcian Blue staining. (A) Fgf8 protein delays chondrogenesis in micromass cultures of chick stage 22–23 limb mesenchyme (n=12). (B) Wnt3a combined with Fgf8 blocks chondrogenesis (n=12). (C) Removal of Fgf8 at day 4 of culture has little effect on chondrogenesis (n=12). (D) Cells treated with Wnt3a and Fgf8 resume chondrogenesis upon removal of the factors at day 4 (n=12). (E–G) Limb mesenchyme cells were expanded for 4 days in presence of Wnt3a alone (E) or in combination with Fgf8 (F,G), trypsinized and replated as micromass cultures. Cells expanded in Wnt3a alone lost their chondrogenic potential (E). Cells expanded in Wnt3a and Fgf8 retained their chondrogenic potential (F), whereas Wnt3a was still able to inhibit their chondrogenesis (G) (n=4). (H) Wnt3a promotes proliferation of limb mesenchyme in micromass cultures, which is enhanced by Fgf8. Fgf8 alone does not enhance proliferation (n=4). (I) Size of limbs cultured 4 days with intact ectoderm, or without ectoderm in presence of the indicated factors (n=8, mean±s.e.m.). (J–N) Representative examples of Alcian Blue-stained limb buds cultured without ectoderm in presence of vehicle (J), Wnt3a (K), Fgf8 (L), Wnt3a and Fgf8 (M), or with ectoderm left intact (N). Scale bar: 500 µm in J–N.

Article Snippet: But in contrast to the effect of Wnt3a alone, the combination of Fgf8 and Wnt3a maintained the undifferentiated state of the cells: following withdrawal of both factors, they retained their ability to differentiate into cartilage ( ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. 3 caption a7 caption a8 Wnt and FGF proteins act in synergy to promote proliferation and maintain the undifferentiated state (A–G) Alcian Blue staining. ( A ) Fgf8 protein delays chondrogenesis in micromass cultures of chick stage 22–23 limb mesenchyme ( n =12). ( B ) Wnt3a combined with Fgf8 blocks chondrogenesis ( n =12). ( C ) Removal of Fgf8 at day 4 of culture has little effect on chondrogenesis ( n =12). ( D ) Cells treated with Wnt3a and Fgf8 resume chondrogenesis upon removal of the factors at day 4 ( n =12). ( E–G ) Limb mesenchyme cells were expanded for 4 days in presence of Wnt3a alone (E) or in combination with Fgf8 (F,G), trypsinized and replated as micromass cultures.

Techniques: Staining, Cell Culture

Journal:

Article Title: Wnt and FGF signals interact to coordinate growth with cell fate specification during limb development

doi: 10.1242/dev.023176

Figure Lengend Snippet: Cells were cultured at high density in the presence of Wnt3a, Fgf8, or both, and samples taken 2, 4 or 6 hours after addition of the factors. Gene expression levels were plotted relative to vehicle controls. Note synergistic regulation of Nmyc, Sdc1 and Sox9 by the combination of Wnt3a and Fgf8 (blue line), and the antagonistic effect of Fgf8 on the induction of Nbl1 by Wnt3a.

Article Snippet: But in contrast to the effect of Wnt3a alone, the combination of Fgf8 and Wnt3a maintained the undifferentiated state of the cells: following withdrawal of both factors, they retained their ability to differentiate into cartilage ( ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. 3 caption a7 caption a8 Wnt and FGF proteins act in synergy to promote proliferation and maintain the undifferentiated state (A–G) Alcian Blue staining. ( A ) Fgf8 protein delays chondrogenesis in micromass cultures of chick stage 22–23 limb mesenchyme ( n =12). ( B ) Wnt3a combined with Fgf8 blocks chondrogenesis ( n =12). ( C ) Removal of Fgf8 at day 4 of culture has little effect on chondrogenesis ( n =12). ( D ) Cells treated with Wnt3a and Fgf8 resume chondrogenesis upon removal of the factors at day 4 ( n =12). ( E–G ) Limb mesenchyme cells were expanded for 4 days in presence of Wnt3a alone (E) or in combination with Fgf8 (F,G), trypsinized and replated as micromass cultures.

Techniques: Cell Culture, Gene Expression

Journal: Oncotarget

Article Title: FGF8 promotes colorectal cancer growth and metastasis by activating YAP1

doi:

Figure Lengend Snippet: QRT-PCR (A) and imunoblot (B) analysis of FGF8 level in human CRC tissues (T) and adjacent normal mucosa tissues (N) from the same patient. All data were from at least three independent experiments. *, P<0.05; **, P<0.01.

Article Snippet: FGF8 recombinant protein was purchased from Protech and used at 250 ng/ml.

Techniques: Quantitative RT-PCR

Journal: Oncotarget

Article Title: FGF8 promotes colorectal cancer growth and metastasis by activating YAP1

doi:

Figure Lengend Snippet: (A) Immunohistochemical staining of FGF8 in tumor and corresponding colorectal mucosa. (B) Immunohistochemical scores for FGF8 in normal colorectal mucosa and CRC tissues. (C) Expression of FGF8 in the primary tumors without (N0) or with (N1/N2) lymph node metastasis was analyzed. Left, overall tumors; middle, stage T1–T2; right, stage T3–T4. (D) Kaplan–Meier survival curves of CRC patients with low (n = 34) and high (n = 38) FGF8 expression. Left, overall tumors; middle, stage T1–T2; right, stage T3–T4. ***, P<0.001.

Article Snippet: FGF8 recombinant protein was purchased from Protech and used at 250 ng/ml.

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Oncotarget

Article Title: FGF8 promotes colorectal cancer growth and metastasis by activating YAP1

doi:

Figure Lengend Snippet: RKO cells were treated with or without FGF8 and/or PD173074. (A) Representative photographs of colony formation 14 days after culture of cells. (B) Proliferation rate as measured by BrdU labeling for 12 hours. (C) Quantitative analysis of cell migration and matrigel invasion assays. Migration was analyzed at 24 h, and invasion at 48 h. (D) Representative phase-contrast images of RKO cell morphology. (E) Expression of Snail, E-cadherin and Vimentin was examined by immunoblot. All data were from at least three independent experiments. **, P<0.01; ***, P<0.001.

Article Snippet: FGF8 recombinant protein was purchased from Protech and used at 250 ng/ml.

Techniques: Labeling, Migration, Expressing, Western Blot

Journal: Oncotarget

Article Title: FGF8 promotes colorectal cancer growth and metastasis by activating YAP1

doi:

Figure Lengend Snippet: (A) Mean tumor volume and Ki67 expression in tumors after subcutaneous transplantation of RKO-FGF8 or RKO-mock cells. (B) Histopathology showing the lung metastases in mice and quantification of the lung weight and number of metastases following tail-vein injection with RKO-FGF8 or RKO-mock cells. **, P<0.01; ***, P<0.001.

Article Snippet: FGF8 recombinant protein was purchased from Protech and used at 250 ng/ml.

Techniques: Expressing, Transplantation Assay, Histopathology, Injection

Journal: Oncotarget

Article Title: FGF8 promotes colorectal cancer growth and metastasis by activating YAP1

doi:

Figure Lengend Snippet: (A) We identified hub proteins implicated in core pathways according to the following four gold standards: degree, link, module and microarray analyses. (B) The global PPI network. (C) The core sub-network in cancer. (D) Identification of FGF8-YAP1 interaction in CRC cells.

Article Snippet: FGF8 recombinant protein was purchased from Protech and used at 250 ng/ml.

Techniques: Microarray

Journal: Oncotarget

Article Title: FGF8 promotes colorectal cancer growth and metastasis by activating YAP1

doi:

Figure Lengend Snippet: (A) Immunoblot analysis of cytoplasmic and nuclear YAP1 in RKO cells treated with or without FGF8 or/and PD173074. β-Actin was used as a cytoplasmic protein loading control, and histone-3 (H3) was used for nuclear protein loading control. (B) MRNA level CTGF and CYR61 was examined by qRT-PCR. (C) Transcription activity of TEAD4 was examined by luciferase assay. (D) Expression of YAP1 and FGF8 in serial human colorectal tumor sections was examined by immunohistochemical staining. (E) Correlation between the expression levels of YAP1and FGF8. (F) Expression of YAP1 in high-FGF8-expressing tumors and low-FGF8-expressing tumors was analyzed. All data were from at least three independent experiments.*, P<0.05; **, P<0.01.

Article Snippet: FGF8 recombinant protein was purchased from Protech and used at 250 ng/ml.

Techniques: Western Blot, Control, Quantitative RT-PCR, Activity Assay, Luciferase, Expressing, Immunohistochemical staining, Staining

Journal: Oncotarget

Article Title: FGF8 promotes colorectal cancer growth and metastasis by activating YAP1

doi:

Figure Lengend Snippet: FGF8-treated RKO cells were transfected with siYAP1 or siNC. (A-B) Proliferative activity was measured by a colony formation assay (A) and BrdU labeling (B). (C) Cell migration and invasion was examined by transwell assay and matrigel invasion assays. (D) Representative phase-contrast images of RKO cell morphology. (E) Expression of Snail, E-cadherin and Vimentin was examined by immunoblot. All data were from at least three independent experiments. *, P<0.05; **, P<0.01; ***, P<0.001.

Article Snippet: FGF8 recombinant protein was purchased from Protech and used at 250 ng/ml.

Techniques: Transfection, Activity Assay, Colony Assay, Labeling, Migration, Transwell Assay, Expressing, Western Blot

Journal: Oncotarget

Article Title: FGF8 promotes colorectal cancer growth and metastasis by activating YAP1

doi:

Figure Lengend Snippet: QRT-PCR analysis of YAP1 mRNA level in FGF8-treated RKO, SW480 and HCT116 cells in the present or absent of PD173074.

Article Snippet: FGF8 recombinant protein was purchased from Protech and used at 250 ng/ml.

Techniques: Quantitative RT-PCR