C/EBPβ directly bound to <i>Ihh</i> promoter.

<p>(A) EMSA for specific binding of C/EBPβ to the <i>Ihh</i> promoter. Consensus oligonucleotide (C), wild-type (WT) and mutant (MT) probes were incubated with nuclear extract from C/EBPβ-transfected ATDC5 cells. Competition and supershift experiments were also performed. Data are representative of two independent experiments performed in duplicate. (B) A ChIP assay for C/EBPβ using ATDC5 cells cultured for 3 weeks. Semi-quantitative RT-PCR was performed using primers as follows: promoter region of <i>Ihh</i> (from −259 to −160) and negative control (from −1274 and −1102 bp). Data are representative of two independent experiments performed in duplicate.</p

C/EBPβ binding element is crucial for RUNX2 to regulate transcriptional activity of <i>Ihh</i>.

<p>(A) Deletion constructs were co-transfected with 0.2 µg of RUNX2 or GFP into HeLa cells. Means ± S.D. of duplicates from three independent experiments are shown. ^*<i>p</i><0.05. (B) Mutation constructs of C/EBPβ and RUNX2 binding elements in pDel3 were co-transfected with 0.2 µg of RUNX2 or GFP into HeLa cells. Means ± S.D. of duplicates from three independent experiments are shown. ^*<i>p</i><0.05. (C) EMSA for specific binding of RUNX2 to the C/EBPβ binding site of <i>Ihh</i> promoter. Wild-type (WT) probe, which harbors C/EBPβ binding site, was incubated with nuclear extract from C/EBPβ-transfected ATDC5 cells. Supershift experiment using RUNX2 antibody was also performed. Data are representative of two independent experiments performed in duplicate. (D) A ChIP assay for RUNX2 using ATDC5 cells cultured for 3 weeks. Semi-quantitative RT-PCR was performed using same primers as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104547#pone-0104547-g005" target="_blank">Figure 5B</a>. Data are representative of two independent experiments performed in duplicate. (E) Immunoprecipitation (IP) and Immunoblotting were performed. Nuclear extract was obtained from C/EBPβ-transfected ATDC5 cells. Immunoprecipitated proteins with C/EBPβ, RUNX2 or IgG antibody were subjected to SDS-PAGE and immunoblotting using C/EBPβ or RUNX2 antibody.</p

C/EBPβ stimulated the expression of Ihh in <i>ex vivo</i> organ cultures.

<p><i>Ex vivo</i> organ culture of tibias dissected from E14.5 mouse embryos. Tibias were cultured for 4 days after transfection with adenovirus vectors expressing LacZ control (top row) and C/EBPβ-LAP (bottom row). Safranin O staining and immunofluorescent staining were performed to localize C/EBPβ, RUNX2 and Ihh. DAPI was used as a counterstain. Red, green and blue bars indicate the proliferative, pre-hypertrophic and hypertrophic zones, respectively. Scale bar, 500 µm. Histological analysis was repeated at least twice for each sample from six pairs of limbs, respectively.</p

C/EBPβ stimulated the expression of <i>Ihh</i> and <i>Runx2</i> in ATDC5 cells.

<p>(A) Western blot of nuclear extracts obtained from ATDC5 cells, which were transfected with LacZ or C/EBPβ-LAP, was performed to investigate the expression of C/EBPβ. Densitometric scanning of C/EBPβ expression was performed. Each density of C/EBPβ was normalized with that of LAMIN A/C and the ratio by corrected densities of C/EBPβ to control on the 4th day was calculated. Data are representative of two independent experiments performed in duplicate. ^*<i>p</i><0.05 vs. LacZ. (B) ATDC5 cells were differentiated for 2 weeks after transfection with adenovirus vectors expressing C/EBPβ-LAP and LacZ control. Expression of <i>Ihh</i> and <i>Pthrp</i> mRNA was determined by real-time RT-PCR. Each value was normalized to <i>18S</i> in the same sample. The value of each mRNA expression relative to that of LacZ on the 4th day was indicated. Means ± S.D. of duplicates from three independent experiments are shown. ^*<i>p</i><0.05 vs. LacZ.</p

C/EBPβ up-regulated transcriptional activity of <i>Ihh</i>.

<p>(A) The <i>Ihh</i> reporter construct containing −1224 to +43 bp of the <i>Ihh</i> promoter, and various deletion constructs were generated. Gray and black boxes indicate RUNX2 binding elements reported by a previous study and C/EBPβ binding motif, respectively. Mutation construct of each element was also generated. (B) The <i>Ihh</i> reporter construct (pFull) was co-transfected with pCMV-LAP and GFP into HeLa cells. Means ± S.D. of duplicates from three independent experiments are shown. (C) The <i>Ihh</i> reporter construct (pFull) was co-transfected with 0.1 µg of pCMV-LAP and various amounts of A-C/EBP into HeLa cells. Means ± S.D. of duplicates from three independent experiments are shown. (D) Deletion constructs were co-transfected with 0.1 µg of pCMV-LAP or GFP into HeLa cells. Means ± S.D. of duplicates from three independent experiments are shown. ^*<i>p</i><0.05. (E) A mutation construct of C/EBPβ binding motif in pDel3 was co-transfected with 0.1 µg of pCMV-LAP or GFP into HeLa cells. Means ± S.D. of duplicates from three independent experiments are shown. ^*<i>p</i><0.05.</p