Luciferase assay The human p57 promoter regions were cloned into the pGL4.10 vector (Promega). Other deletion constructs were created by the PCR technique. Tandem-repeat constructs were created by ligating the double strand oligonucleotides from 2150 to 2130 bp into pGL4.10 vector. Transfection in HuH-7 and ATDC5 cells was performed in quadruplicate using Fugene (Roche). For PTH or forskolin stimulation, cells were cultured with PTH (10 nM) or forskolin (10 nM) at the time of transfection. The luciferase assay was performed with a PicaGene Dual SeaPansy Luminescence Kit (Toyo Ink) and GloMaxTM 96 Microplate Luminometer (Promega). Electrophoretic Mobility Shift Assay (EMSA) Nuclear extracts were prepared from ATDC5 cells adenovirally transfected with C/EBPb. Oligonucleotide probes of the 2160 to 2120 bp region sequence in the p57 promoter were labeled with digoxigenin by using a DIG gel shift kit (Roche). For competition analyses, 50-fold excess of unlabeled competitor probe was included in the binding reaction. For the supershift experiments, 1 mL of an antibody to C/EBPb (Santa Cruz Biotechnology Inc.) was added. Microarray analysis Total RNA was isolated from C3H10T1/2 cells with retroviral introduction of C/EBPb or the empty vector after 1 week of culture. The microarray experiment was performed using the Gene Chip Mouse Genome 430 2.0 Array (Affymetrix), scanned by GeneChip Scanner 3000, and analyzed using GCOS ver 1.4 software. Immunoblotting ATDC5 cells were cultured with PTH (10 nM) or forskolin (10 nM) for 0 to 30 min, and then their cytoplasmic and nuclear proteins were extracted with an NE-PER (Pierce Chemical). For immunoblot analysis, lysates were fractionated by SDS-PAGE and transferred onto nitrocellulose membranes (BIO-RAD). The membranes were incubated with an antibody to C/EBPb (Santa Cruz), or an antibody to actin (Sigma). Immunoreactive bands were visualized with ECL Plus (Amersham Biosciences). Osteoarthritis experiment The surgical procedure to create an osteoarthritis experimental model was performed on 8-week-old male mice, as we have reported previously [17,20,21]. At the indicated time points after surgery, the mice were killed, and the entire knee joints were dissected and fixed for 24 h at 4uC in 4% PFA. The specimens were decalcified for 2 weeks with 10% EDTA (pH 7.4) at 4uC and sectioned in 5-mm slices. Sections were stained with Safranin O– fast green. Destruction of cartilage was quantified according to the OARSI grading system [22]. For immunohistochemistry, the sections were incubated with antibodies to C/EBPb (Santa Cruz), COL10 (LSL) or the nonimmune rabbit IgG as the negative control diluted 1:500 in blocking reagent, and the localization was detected with HRP-conjugated secondary antibody (Promega). Statistical analysis Means of groups were compared by ANOVA, and significance of differences was determined by post-hoc testing using Bonferro- ni’s method. References C/EBPb in Chondrocytes Supporting Information Table S1 Microarray analysis shows the changes in expression of cell cycle factors by C/EBPb overexpression. Ratios of mRNA levels in C3H10T1/2 cells with retroviral introduction of C/EBPb in comparison with the control empty vector were determined by Gene Chip Mouse Genome 430 2.0 Array (Affymetrix). All results of the microarray analysis are provided at ArrayExpress (accession number: E-MEXP-1984). Found at: doi:10.1371/journal.pone.0004543.s001 (0.02 MB PDF) Figure S1 C/EBPd shows similar expression and function in chondrocytes to those of C/EBPb. (A) Immunostaining with an antibody to C/EBPd in the tibial cartilage of wild-type (WT) and C/EBPb2/2 littermates (E16.5). Red, blue, and green bars indicate layers of proliferative zone, hypertrophic zone, and bone area, respectively. Scale bars, 100 mm. (B) Relative mRNA levels of COL10, MMP13, and VEGF of ATDC5 cells with retroviral transfection of C/EBPd or the control GFP determined by real- time RT-PCR at 2 weeks of culture after confluency. (C) The p57 promoter activity in ATDC5 cells transfected with luciferase- reporter construct containing the 59-flanking sequences from 2150 to +226 bp of the p57 promoter with effector plasmid expressing C/EBPd, C/EBPb, or the control GFP. All data are expressed as means (symbols or bars)6SEM (error bars) of 6 wells per group. *P,0.01 vs. GFP. Found at: doi:10.1371/journal.pone.0004543.s002 (0.68 MB PDF) Figure S2 PTH and forskolin have no effects on C/EBPb protein level and p57 promoter activity. (A) Time course of C/ EBPb protein level in cultured ATDC5 cells. After the indicated time of treatment with PTH (10 nM) and forskolin (10 nM), the C/EBPb protein levels in the cytoplasmic fraction (C) and nuclear fraction (N) were determined by immunoblotting with an antibody to C/EBPb or actin as the loading control. (B) Effects of PTH, forskolin or the control on HuH-7 cells transfected with luciferase- reporter construct containing the 59-flanking fragment (2150 to +226 bp) of the p57 promoter with effector plasmid expressing C/ EBPb or GFP as the control. The promoter activity was determined by the luciferase assay after 2 d of treatment. Found at: doi:10.1371/journal.pone.0004543.s003 (0.07 MB PDF) Figure S3 Effects of cGKII and GSK-3b overexpression on p57 promoter activity. The promoter activity was determined by the luciferase assay in HuH-7 cells transfected with luciferase-reporter construct containing the 59-flanking fragment (2150 to +226 bp) of the p57 promoter with effector plasmid expressing C/EBPb or GFP as the control. Found at: doi:10.1371/journal.pone.0004543.s004 (0.02 MB PDF) Author Contributions Conceived and designed the experiments: MH FK TI KN UiC HK. Performed the experiments: MH FK AF SO TO. Analyzed the data: MH FK AF. Contributed reagents/materials/analysis tools: SO NK TO YK TS FY TI UiC. Wrote the paper: MH NK HK. 1. Kronenberg HM (2003) Developmental regulation of the growth plate. Nature 423: 332–336. 2. Hunziker EB (1994) Mechanism of longitudinal bone growth and its regulation by growth plate chondrocytes. Microsc Res Tech 28: 505–519. PLoS ONE | www.plosone.org 11 February 2009 | Volume 4 | Issue 2 | e4543