Journal: PLoS ONE

Article Title: Activation of FGF Signaling Mediates Proliferative and Osteogenic Differences between Neural Crest Derived Frontal and Mesoderm Parietal Derived Bone

doi: 10.1371/journal.pone.0014033

Figure Lengend Snippet: Endogenous levels of FGF-2 were analyzed on FOb and POb cells by immunoblotting using antiFGF-2 antibody. Higher levels of high molecular weight (HMW) and low molecular weight (LMW) FGF-2 forms were observed in FOb compared to POb at all stages analyzed. The total cellular FGF-2 proteins were quantified by densitometry (lower panel), α-tubulin was used as loading control. Histogram represents the densitometric analysis of electrophoresis bands, the relative intensities of bands were normalized to their respective loading control and set as 100% The results are presented as the mean ± SD of three independent experiments. The asterisks represent P value <0.001.

Article Snippet: A polyclonal goat anti-FGF-2 antibody (R&D systems, MN) was used for FGF-2 neutralization.

Techniques: Western Blot, Molecular Weight, Electrophoresis

Journal: PLoS ONE

Article Title: Activation of FGF Signaling Mediates Proliferative and Osteogenic Differences between Neural Crest Derived Frontal and Mesoderm Parietal Derived Bone

doi: 10.1371/journal.pone.0014033

Figure Lengend Snippet: Cell proliferation of FOb and POb harvested from E17.5 mice was measured by BrdU. Osteoblast cells were cultured with conditioned serum-free media collected from frontal osteoblasts (FCM) or parietal osteoblasts (PCM) after 24 hours culture. The conditioned media were incubated with neutralizing anti-FGF-2 antibody at different concentrations (1, 2 and 4 µg/ml). Same concentrations of irrelevant rabbit IgG were used as control. 20 ng/ml of hrFGF-2 was added to the medium as a positive control (SF + FGF-2) and serum-free medium as a negative control (SF). FCM induced significant mitogenic effect, as much as the exogenous added rhFGF-2, on both FOb and POB cells. Anti-FGF-2 antibody blocked the FGF-2 mitogenic effect in a dose-dependent manner. Conversely PCM did not elicit mitogenic effect either on FOb or POb cells and there was no blocking effect by the FGF-2 neutralizing antibody on PCM.

Article Snippet: A polyclonal goat anti-FGF-2 antibody (R&D systems, MN) was used for FGF-2 neutralization.

Techniques: Cell Culture, Incubation, Positive Control, Negative Control, Blocking Assay

Journal: PLoS ONE

Article Title: Activation of FGF Signaling Mediates Proliferative and Osteogenic Differences between Neural Crest Derived Frontal and Mesoderm Parietal Derived Bone

doi: 10.1371/journal.pone.0014033

Figure Lengend Snippet: FOb and POb cells harvested from E17.5 mice were cultured in serum free α-MEM for 12 hours in presence of specific inhibitors of the FGF signaling pathways to pre-empty endogenous FGF-2 activity. Then cells were treated with 20 ng/ml of rhFGF-2. A , effect of exogenous FGF-2 and inhibitors of MAPK signaling pathway. FGF-2 stimulation for 30 minutes increased phosphorylation of ERK protein equally in FOb and POb cells. Co-treatment with 10 µM U0126 inhibitor inhibited the FGF-2 induction as well as the endogenous phosphorylation of pERK protein in untreated FGF-2 FOb and POb cells. Histogram represents densitometric analysis of electrophoresis bands as above. B , effect of exogenous FGF-2 and inhibitors of PI3K signaling pathway. FGF-2 treatment resulted in increased pAkt phosphorylation which was two fold higher in POb cells compared to FOb cells. Treatment with 20 µM LY294002 inhibitor mostly abrogated the effect of exogenous FGF-2 and drastically reduced the endogenous level of pAkt protein in untreated FGF-2 FOb cells. Histogram represents densitometric analysis of electrophoresis bands. C , effect of exogenous FGF-2 and inhibitors of PKC α/β signaling pathway. FGF-2 treatment also induced an increased phosphorylation of PKC α/β proteins which was inhibited by co-treatment with 2 µM GÖ6983 inhibitor. Treatment with the inhibitor also reduced the endogenous level of p PKC α/β proteins in untreated FGF-2 cells. Histogram represents densitometric analysis of electrophoresis bands. D , effect of exogenous FGF-2 and inhibitors of PKC δ signaling pathway. FGF-2 and/or inhibitor treatments on PKC δ protein produced a similar effect to that observed on PKC α/β proteins. Asterisk * represents statistical significance (*p<0.05).

Article Snippet: A polyclonal goat anti-FGF-2 antibody (R&D systems, MN) was used for FGF-2 neutralization.

Techniques: Cell Culture, Activity Assay, Electrophoresis, Produced

Journal:

Article Title: Phenotypic consequences of lung-specific inducible expression of FGF-3

doi: 10.1073/pnas.101116598

Figure Lengend Snippet: (A) RPA of FGF-3 and GLp65 expression in the lungs after administration of RU486 at a dosage of 500 μg/kg for 1 week (+) on transgenic lines 1292, 1138H, and 1137. Bitransgenic littermates were administered placebo pellets as control (−). RPA of FGF-3 mRNA (B) and Western blot analysis of FGF-3 protein expression (C) in the lungs of mice from line 1138H after administration of placebo or RU486 at a dosage of 66, 130, and 200 μg/kg for 1 month are shown. Each lane represents an independent mRNA and protein from an individual mouse.

Article Snippet: The membranes then were probed with goat anti-mouse FGF-3 polyclonal IgG (Santa Cruz Biotechnology) and horseradish peroxidase-conjugated anti-goat IgG (Santa Cruz Biotechnology).

Techniques: Expressing, Transgenic Assay, Western Blot

Journal:

Article Title: Phenotypic consequences of lung-specific inducible expression of FGF-3

doi: 10.1073/pnas.101116598

Figure Lengend Snippet: Histological changes in the lungs of GLp65/FGF-3 bitransgenic mice treated with placebo or 500 μg/kg RU486. (A) H&E-stained section of lung from placebo-treated mouse. (B) Immunohistochemical staining for Mac-3 of lung from 1137 bitransgenic mouse treated with RU486 for 6 weeks. Free alveolar macrophages (FAMs) are abundant in the alveoli as indicated by the arrow. (C) Immunohistochemical staining for Nkx2.1 of lung from the 1138H bitransgenic mouse treated with 500 μg/kg RU486 for 2 weeks. Type II epithelial cells are increased strikingly along the septa and in alveoli as indicated by the arrow. (D) H&E section of lung from the 1292 bitransgenic mouse treated with 500 μg/kg RU486 for 1 week.

Article Snippet: The membranes then were probed with goat anti-mouse FGF-3 polyclonal IgG (Santa Cruz Biotechnology) and horseradish peroxidase-conjugated anti-goat IgG (Santa Cruz Biotechnology).

Techniques: Staining, Immunohistochemical staining

Journal:

Article Title: Phenotypic consequences of lung-specific inducible expression of FGF-3

doi: 10.1073/pnas.101116598

Figure Lengend Snippet: The response of 1138H bitransgenic mouse lungs to the induction of FGF-3 for 4 weeks with varying doses of RU486. (A) H&E-stained section of lung from placebo-treated 1138H bitransgenic mouse. (B) H&E-stained section of lungs from 1138H bitransgenic mouse treated with 130 μg/kg RU486. FAMs are abundant. (C) H&E-stained section of lungs from 1138H bitransgenic mouse treated with 200 μg/kg RU486. Type II cell hyperplasia is apparent.

Article Snippet: The membranes then were probed with goat anti-mouse FGF-3 polyclonal IgG (Santa Cruz Biotechnology) and horseradish peroxidase-conjugated anti-goat IgG (Santa Cruz Biotechnology).

Techniques: Staining

Journal:

Article Title: Phenotypic consequences of lung-specific inducible expression of FGF-3

doi: 10.1073/pnas.101116598

Figure Lengend Snippet: RPA of FGF-3 (A) and cyclin mRNAs (B) in the lungs of 1138H bitransgenic mouse lungs after the administration and withdrawal of 500 μg/kg RU486. −, RNA from mice treated with placebo; On 1 wk, mice treated with RU486 for 1 week; On 2 wks, mice treated with RU486 for 2 weeks; Off 1 wk, mice after withdrawal of RU486 for 1 week; Off 2 wks, mice after the withdrawal of RU486 for 2 weeks after treatment with RU486; Re-on 2 wks, mice treated with RU486 after withdrawal of RU486 for 2 weeks. Expression of cyclophillin (cph) served as a control for RNA-loading analysis of FGF-3 expression. Expression of L32 served as a control for RNA loading for cyclin gene expression (data not shown). Each lane represents an independent mRNA from an individual mouse.

Article Snippet: The membranes then were probed with goat anti-mouse FGF-3 polyclonal IgG (Santa Cruz Biotechnology) and horseradish peroxidase-conjugated anti-goat IgG (Santa Cruz Biotechnology).

Techniques: Expressing

Journal:

Article Title: Phenotypic consequences of lung-specific inducible expression of FGF-3

doi: 10.1073/pnas.101116598

Figure Lengend Snippet: The effect of FGF-3 induction on IL-7, CM-CSF, and pulmonary epithelial marker gene expression. (A) RPA of the expression of IL-7 and GM-CSF in lungs from 1137 mice treated with 500 μg/kg RU486 for either 1 or 6 weeks. L32 was used as internal control for RNA loading in the RPA. (B) S1 nuclease protection analysis of the expression of epithelial markers, SP-A, SP-B, and SP-C, CCSP, and cytochrome p450 2F2 in bitransgenic mice from line 1138H were administered RU486 for 1 month at a dose of 0, 66, 130, and 200 μg/kg. L32 was used as internal control for RNA loading in the S1 nuclease protection analysis. Control samples were assayed in triplicate (data not shown).

Article Snippet: The membranes then were probed with goat anti-mouse FGF-3 polyclonal IgG (Santa Cruz Biotechnology) and horseradish peroxidase-conjugated anti-goat IgG (Santa Cruz Biotechnology).

Techniques: Marker, Expressing