FGF4 increases proliferation of mESCs.

<p>mESCs were incubated in the presence of 0 to 200 ng/ml FGF4 in 96-multiwell plates or with the indicated concentration of FGF4 in 6-well culture plates for 48 h and then further processed for BrdU incorporation assay (A) and hematoxylin staining (B), respectively. Experimental results shown in (A) represent the mean ± SD from three separate experiments. **p<0.01 indicates a significant difference between experiments and untreated control cells.</p

Effects of anti-FGF4 antibody on FGF4-induced cell proliferation and protein expression associated with cell cycle progression.

<p>mESCs cultured in 6-well culture plates were incubated in the presence of 50 ng/ml FGF4 and/or 100 ng/ml anti-FGF4 antibody and then proliferation rates were analyzed using ³H-TdR incorporation (A, left panel) and Cell Counting Kit-8 (A, right panel) 48 h after treatment. *p<0.05, **p<0.01, and ***p<0.001 indicate significant differences between experiments. (B) Expression patterns of cell cycle regulatory proteins 24 h after treatment were analyzed by Western blotting. (C) Data from Western blot analysis were quantified by densitometry after normalizing bands to α-tubulin levels. **p<0.01 and ***p<0.001 vs. untreated cells. ^##p<0.01 and ^###p<0.001 vs. cells treated with FGF4 only. (D) mESCs cultured under the same conditions as (C) were also processed for analysis of PCNA expression by flow cytometry. a-F4, anti-FGF4 antibody.</p

Exogenous FGF4 decreases osteogenic differentiation of mESCs.

<p>(A) mESCs were incubated in the presence of DAG and/or 50 ng/ml FGF4. After 5 days of incubation, DNA synthesis (left panel) and cell proliferation (right panel) rates were determined. At the same time, cells were processed for Alizarin red staining (B) and absorbance of the dye (C) was also determined at 560 nm. (D) Runx2 protein in mESCs cultured with DAG and/or 50 ng/ml FGF4 for 5 days was analyzed by flow cytometry. (E) ALP activity in the cells was measured after 3 and 5 days of differentiation. *p<0.05, **p<0.01 and ***p<0.001 vs. untreated control cells. ^#p<0.05 vs. cells treated with DAG only.</p

Addition of FGF4 diminishes DAG-induced expression of Runx2 and osterix in mESCs.

<p>Cells were incubated in the presence or absence of DAG and 50 ng/ml FGF4 for 24 h, and expression patterns of bone-specific genes were analyzed by real-time RT-PCR. **p<0.01 and ***p<0.001 vs. untreated control cells. ^#p<0.05 vs. cells treated with DAG only.</p

FGF4 stimulates proliferation and mineralization of hPDLSCs.

<p>(A) hPDLSCs at passage 3 were labeled with the indicated primary antibodies and then analyzed by flow cytometer. The cells were incubated for 48 h with 50 ng/ml FGF4 and/or 100 ng/ml (B), or with each of MAPK inhibitors (C) in 96-multiwell plates. The cells were processed for cell proliferation assay. **p<0.01 vs. GM alone. ^#p<0.05 vs. FGF4 treatment alone. (D) hPDLSCs were incubated in the presence of DAG with and without 50 ng/ml FGF4 and 100 ng/ml anti-FGF4 for 14 days and then were processed for Alizarin red staining. (E) Absorbance of the dye was determined at 560 nm. hPDLSCs were also processed for analysis of ALP activity (F) and mRNA expression (G) after 5 days of differentiation. *p<0.05 vs. DAG alone. F4, FGF4; GM, growth medium; NC, non-significant.</p

JNK acts as an upstream mediator of c-Jun phosphorylation and AP-1 activation.

<p>(A) mESCs transfected with siJNK were incubated in the presence or absence of 50 ng/ml FGF4 and then processed for Western blot analysis 1 h after treatment. (B) Data were quantified from triplicate experiments by densitometry after normalizing bands to total c-Jun protein. mESCs were also treated with each MAPK-specific inhibitor (10 µM) 30 min before addition of 50 ng/ml FGF4, and cells were adjusted to analyze AP-1-DNA binding activity by EMSA (C) or AP-1 activity using a luciferase assay kit (D) after 2 h of incubation. *p<0.05 and ***p<0.001 vs. untreated controls. ^###p<0.001 vs. cells treated with 50 ng/ml FGF4 only.</p

Inhibition of JNK suppresses FGF4-mediated proliferation of mESCs.

<p>(A) mESCs were pretreated with MAPK inhibitor or anti-FGF4 antibody 30 min before addition of 50 ng/ml recombinant FGF4, and cell proliferation rate was determined 48 h after treatment using a cell proliferation assay kit. In addition, siRNAs directed against JNK or GFP were transfected into mESCs. At 24 h post-transfection, cells were exposed to 50 ng/ml FGF4 and cell proliferation rate (B) and ³H-TdR incorporation (C) were measured 48 h after treatment. *p<0.05, **p<0.01, and ***p<0.001 vs. untreated cells. ^##p<0.01 and ^###p<0.001 vs. cells treated with FGF4 only.</p

Addition of FGF4 activates JNK and ERK, but not p38 kinase, in mESCs.

<p>mESCs cultured in 6-well culture plates were incubated in the presence of 50 ng/ml FGF4 and/or 100 ng/ml anti-FGF4 antibody for 1 h and then processed to determine levels of phosphorylated MAPK by immunoblotting (A) or by immunometric analyses (B). **p<0.01 and ***p<0.001 indicate significant differences between experiments.</p

The primer sequences used to analyze the expression of bone-specific genes.

<p>The primer sequences used to analyze the expression of bone-specific genes.</p