Journal:

Article Title: Activin is an essential early mesenchymal signal in tooth development that is required for patterning of the murine dentition

doi:

Figure Lengend Snippet: FGF8 is required to maintain expression of activin βA expression in the odontogenic mesenchyme. (A,B) Serial frontal sections through the prospective molar region of an E11.5 wild-type showing the complementary expression patterns of activin βA in the mesenchyme (A) and Fgf8 in the epithelium (B) on consecutive sections. The tooth germ epithelium is outlined in red. (C,D) activin βA expression in E11.5 mandible explants that were cultured for 2 days with (C) and without (D) epithelium. The arrows indicate the positions of incisor buds. (E,F) activin βA expression in E11.5 mandible explants that were cultured for 1 day without epithelium but in the presence of implanted FGF8 beads (E) or BSA control beads (F). (G,H) DIG whole-mount in situ hybridization of E11.0 mandibles that were cultured without epithelium but in the presence of beads. (G) FGF8 maintains activin βA expression in the mesenchyme. Beads were placed on half of the mandible. (H) BMP4 does not inhibit the maintenance activity of FGF8. The experiment was performed on both halves of the mandible.

Article Snippet: For the application of FGF8 protein, heparin acrylic beads (Sigma) were washed and then incubated overnight in 1 mg/ml FGF8 protein (recombinant mouse FGF8b; R&D Systems, Abingdon, UK) at 4°C.

Techniques: Expressing, Cell Culture, In Situ Hybridization, Activity Assay

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: Journal of Biological Chemistry

Article Title: Stem Cell Factor/c-kit Signaling Promotes the Survival, Migration, and Capillary Tube Formation of Human Umbilical Vein Endothelial Cells

doi: 10.1074/jbc.m311643200

Figure Lengend Snippet: FIG. 2. Effects of neutralizing antibodies (Ab) against VEGF, bFGF, and HGF on SCF-driven capillary tube formation of HU- VECs. HUVECs were three-dimensionally cultured for 3 days in type I collagen gel containing the assay medium with SCF or the indicated angiogenic factors and increasing amounts of antibodies against them. Cells were stained with MTT, and the total length of tube structures per field was measured as described under ‘‘Experimental Procedures.’’ All experiments were done at least in duplicate and repeated three times.

Article Snippet: Recombinant human VEGF was purchased from Genzyme Tech (Minneapolis, MN).

Techniques: Cell Culture, Staining

Journal: Journal of Biological Chemistry

Article Title: Stem Cell Factor/c-kit Signaling Promotes the Survival, Migration, and Capillary Tube Formation of Human Umbilical Vein Endothelial Cells

doi: 10.1074/jbc.m311643200

Figure Lengend Snippet: FIG. 1. Capillary tube formation of HUVECs driven by SCF and other angiogenic factors. HUVECs were three-dimensionally cultured for 3 days in type I collagen gel containing the assay medium with SCF or indicated angiogenic factors, and the resulting pictures of capillary tubes were observed with phase contrast microscopy. Cells were stained with MTT, and the total length of tube structures per field was measured as described under ‘‘Experimental Procedures.’’ A, upper, MTT-stained capillary tube morphology (magnification, 40); lower, nonstained capillary tube morphology (magnification, 100). A panels, no addition; b panels, SCF (100 ng/ml); c panels, VEGF (20 ng/ml); d panels, FGF-2 (20 ng/ml); f panels, HGF (20 ng/ml) (magnification, 100) B, dose dependence of SCF. All of experiments were done at least in duplicate and repeated three times.

Article Snippet: Recombinant human VEGF was purchased from Genzyme Tech (Minneapolis, MN).

Techniques: Cell Culture, Microscopy, Staining

Journal: Journal of Biological Chemistry

Article Title: Stem Cell Factor/c-kit Signaling Promotes the Survival, Migration, and Capillary Tube Formation of Human Umbilical Vein Endothelial Cells

doi: 10.1074/jbc.m311643200

Figure Lengend Snippet: FIG. 3. Endothelial survival and mi- gration driven by SCF. A, HUVECs were cultured in the assay medium with various amounts of SCF or VEGF for 72 h on 96-well plates coated with collagen type I. The number of viable cells were counted by WST-1 as described under ‘‘Experimental Procedures.’’ B, HUVECs were cultured to confluence in the assay medium containing SCF or the indicated angiogenic factors on 12-well plates coated with collagen type I. Then, conflu- ent monolayers were wounded with a pi- pette chip and further incubated in the same medium. After 24 h, the cells that had migrated across the edge of the wound were observed as described under ‘‘Experimental Procedures’’ (magnifica- tion, 100). All of experiments were done at least in duplicate and repeated three times.

Article Snippet: Recombinant human VEGF was purchased from Genzyme Tech (Minneapolis, MN).

Techniques: Cell Culture, Incubation

Journal: Journal of Biological Chemistry

Article Title: Stem Cell Factor/c-kit Signaling Promotes the Survival, Migration, and Capillary Tube Formation of Human Umbilical Vein Endothelial Cells

doi: 10.1074/jbc.m311643200

Figure Lengend Snippet: FIG. 5. Effects of STI-571 (c-kit tyro- sine kinase inhibitor) on SCF-in- duced phenotypes of HUVECs. A, ef- fect of STI-571 on SCF or VEGF driven capillary tube formation. HUVECs were three-dimensionally cultured for 3 days in type I collagen gels containing the assay medium with SCF or VEGF and increas- ing amounts of STI-571. B, effects of STI- 571 on SCF- or VEGF-driven cell sur- vival. HUVECs were cultured for 3 days in the assay medium with SCF or VEGF and increasing amounts of STI-571 on 96- well plates coated with collagen type I. C, effects of STI-571 on SCF-driven phos- phorylation of c-kit, Erk1/2, and Akt. Con- fluent monolayers of HUVECs were cul- tured in the assay medium containing 0.5% FBS for 24 h on dishes coated with collagen type I. Cells were then treated with increasing amounts of STI-571 for 60 min, followed by SCF stimulation (100 ng/ ml) for 10 min. Cells were lysed and ana- lyzed by Western blotting. All of experi- ments were done at least in duplicate and repeated three times.

Article Snippet: Recombinant human VEGF was purchased from Genzyme Tech (Minneapolis, MN).

Techniques: Cell Culture, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Stem Cell Factor/c-kit Signaling Promotes the Survival, Migration, and Capillary Tube Formation of Human Umbilical Vein Endothelial Cells

doi: 10.1074/jbc.m311643200

Figure Lengend Snippet: FIG. 6. Comparison of SCF- and VEGF-induced tyrosine phosphorylation of signal molecules. Confluent monolayers of HUVECs were cultured in the assay medium containing 0.5% FBS for 24 h on dishes coated with collagen type I. Cells were treated with the indicated amounts of SCF or VEGF for 10 min, and lysed cells were analyzed by Western blotting.

Article Snippet: Recombinant human VEGF was purchased from Genzyme Tech (Minneapolis, MN).

Techniques: Comparison, Phospho-proteomics, Cell Culture, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Stem Cell Factor/c-kit Signaling Promotes the Survival, Migration, and Capillary Tube Formation of Human Umbilical Vein Endothelial Cells

doi: 10.1074/jbc.m311643200

Figure Lengend Snippet: FIG. 7. Effects of wortmannin, LY294002, or PD98059 against SCF- or VEGF-induced phenotypes of HUVECs. A–C, effect of wortmannin, LY294002, or PD98059 on SCF- or VEGF-driven capillary tube formation. HUVECs were three-dimensionally cultured for 3 days in type I collagen gels containing the assay medium with SCF or VEGF and increasing amounts of wortmannin (A), LY294002 (B), or PD98059 (C). D, effect of wortmannin, LY294002, or PD98059 on SCF- or VEGF-driven phosphorylation of c-kit/KDR, Erk1/2, and Akt. Confluent monolayers of HUVECs were cultured in the assay medium containing 0.5% FBS for 24 h on dishes coated with collagen type I. Cells were then treated with the indicated concentrations of wortmannin, LY294002, or PD98059 for 60 min, followed by SCF (100 ng/ml) or VEGF (20 ng/ml) stimulation for 10 min. Cells were lysed and analyzed by Western blotting. All of the experiments were done at least in duplicate and repeated three times.

Article Snippet: Recombinant human VEGF was purchased from Genzyme Tech (Minneapolis, MN).

Techniques: Cell Culture, Phospho-proteomics, Western Blot