Cell culture and RT-PCR
C3H10T1/2 cells (American Type Culture Collection (ATCC), USA) were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 µg/ml) under 5% CO2 at 37°C. Total RNA was extracted using the SV Total RNA Isolation System (Promega, Madison, WI, USA), and 1.5 µg of total RNA was converted to cDNA using the GeneAmp® Gold RNA PCR Core Kit (Applied Biosystems, Foster City, CA, USA) in accordance with the manufacturer's instructions. The cDNA was advanced to PCR amplification using Platinum® Blue PCR SuperMix (Invitrogen Life Technologies Corp., Carlsbad, CA, USA) and primer sets specific for Opn, Col11a2, Ppar-γ, and β-actin genes. Primer sequences are summarized in . Each reaction mixture contained 1 µl of cDNA, 20 µl of Platinum® Blue PCR SuperMix, and 10 pmol of each primer. Denaturation for 2 min at 95°C was followed by 30 cycles of 30 s at 95°C, 30 s at 60°C, and 30 s at 72°C, and a final extension of 7 min at 72°C. Aliquots of the PCR products were electrophoresed on 2% agarose gels, visualized by ethidium bromide staining, and directly sequenced using the ABI PRISM® BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) to confirm correct amplification in each reaction.
Primer Sets Used for RT-PCR.
The pGL3-Opn promoter construct was a gift from Dr. Piia Aarnisalo, Helsinki, Finland 
. The pGL3-Col11a2 promoter construct has been described previously 
. The pGL3-Ppar-γ2 promoter construct containing tandem repeats of C/EBP-binding sites and its deletion mutants, pGL3-334 (lacking the distal C/EBP-binding site) and pGL3-320 (lacking both C/EBP-binding sites), were gifts from Dr. Xu Cao, Birmingham, AL, USA 
. Variants of pGL3-Opn (pGL3-Opn mut) and pGL3-Col11a2 (pGL3-Col11a2 mut) were created by mutating potential C/EBP-binding sites using the Gene Editor™ Site-Directed Mutagenesis kit (Promega), and successful mutagenesis was confirmed by DNA sequencing. Primer sequences for mutagenesis will be forwarded on request.
Full-length EWSR1-DDIT3 cDNA, in which exon 7 of EWSR1 was in-frame fused to exon 2 of DDIT3 with a serine (AGT) to methionine (ATG) transition at the junction, was amplified by RT-PCR from cDNA of MLS samples collected from the left thigh of a 19-year-old female 
using the forward primer EWSR1 exon 1F 5′-aatggcgtccacggattacagtacc-3′ and reverse primer DDIT3 exon 4R 5′-tcatgcttaatacagattcaccattcg-3′. The products were cloned into a pCR®2.1-TOPO® vector using the TOPO TA Cloning® Kit (Invitrogen Life Technologies Corp.), and the correct sequences were confirmed by DNA sequencing. The EcoRI fragment containing full-length EWSR1-DDIT3 cDNA from correct clones was in-frame inserted into the EcoRI site of the pFLAG-CMV4 mammalian expression vector (Sigma) to produce pFLG-CMV4 EWSR1-DDIT3. Full-length EWSR1 cDNA was amplified from human placenta using the forward primer EWSR1 exon 1F and reverse primer EWSR1 exon 17R 5′-ctagtagggccgatct ctgcgctc-3′. Full-length DDIT3 cDNA was amplified from the abovementioned MLS sample using the forward primer DDIT3 exon 2F 5 ′-atgttcaagaaggaagtgtatcttc-3′ and reverse primer DDIT3 exon 4R. The cDNAs were similarly processed to produce pFLAG-CMV4 EWSR1 and pFLAG-CMV4 DDIT3 expression vectors. The resultant proteins were FLAG epitope tagged at the N termini. Two forms of mutant EWSR1-DDIT3 expression vectors were generated by mutating pFLG-CMV4 EWSR1-DDIT3 using the Gene Editor™ Site-Directed Mutagenesis kit (Promega), and successful mutagenesis was confirmed by DNA sequencing. pFLAG-CMV4 EWSR1-DDIT3 del LZ contained a stop codon just 5′ to the codon for the first leucine residue of the dimer forming LZ domain, so that the entire LZ domain, composed of 38 C-terminal amino acid residues, was deleted. Regarding pFLAG-CMV4 EWSR1-DDIT3 mut LZ, all five codons for leucine residues in the LZ domain were changed to codons for glycine residues. Each correct FLAG-tagged protein expression after transient transfection was confirmed by Western blotting using monoclonal anti-FLAG® M2 (Clone M2) antibody (F3165; Sigma) ().
Protein expression by each FLAG-tagged expression vector.
Transfection and luciferase assay
Transient transfection experiments were performed in C3H10T1/2 cells seeded on 6-well plates using X-tremeGENE 9 reagent (Roche Molecular Biochemicals, Indianapolis, IN, USA) with 875 ng pGL3 reporter plasmid, 125 ng pFLAG-CMV4 expression vector, and 30 ng pRL-TK Renilla internal control plasmid (Promega), according to the manufacturer's instructions. For luciferase assays, the cells were harvested 48 h after transfection and luciferase activity was monitored using the Dual Luciferase® Reporter Assay System (Promega) on a TD-20/20 Luminometer (Turner Designs, Sunnyvale, CA, USA). Transfection in duplicate was repeated at least three times, and the luciferase activity was normalized to internal controls. The results are shown as average ± standard errors. For inhibition of HDACs or DNA methytransferases, transfected cells were treated with 100 ng/ml TSA (Sigma) or 5 µM AZA (Sigma), respectively, 24 h before the luciferase assay and compared with transfected cells without TSA or AZA treatment.
Cells transfected with each pFLAG-CMV4 expression vector in a 10-cm dish were lysed with 1 ml of buffer A (10 mM Tris, pH 7.5, 100 mM NaCl, 2.5 mM MgCl2, 0.5% Triton X-100, and 10 mM dithiothreitol (DTT)) supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail (Sigma). Twenty microliters of 2× SDS sample buffer and 4 µl of DTT were added to 20 µl of the cell lysate. The sample mixtures were denatured for 5 min at 95°C, separated by SDS-PAGE using a 5–15% gradient gel (Bio-Rad Laboratories, Hercules, CA, USA), transblotted onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad Laboratories), and subjected to Western blotting using monoclonal anti-FLAG® M2 (Clone M2) antibody (F3165; Sigma). Protein bands were visualized on an X-ray film using the ECL Western blotting detection system (GE Healthcare, Buckinghamshire, UK) ().
Transient ChIP assay
This is a well-accepted technique developed to study in vivo
binding of transcription factors to specific sites on promoters of reporter constructs 
. The ChIP-IT™ Express Enzymatic kit (Active Motif, Carlsbad, CA, USA) was used according to the manufacturer's instructions with minor modifications. In brief, C3H10T1/2 cells plated on three 10-cm dishes were transiently transfected with 5 µg pGL3 reporter plasmid together with 5 µg pFLAG-CMV4 expression vector per dish. The cells were maintained for 48 h and cross-linked with 1% formaldehyde for 5 min at room temperature. Soluble nuclear material containing reporter plasmids was collected, enzymatically sheared for 10 min at 37°C, and then immunoprecipitated with 3 µg antibody against N-terminal FLAG epitope tags (F3165; Sigma), C/EBPα (14AA, sc-61X; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), C/EBPβ (C-19, sc-150X; Santa Cruz Biotechnology Inc.), HDAC1 (C-19, sc-6298X; Santa Cruz Biotechnology Inc.), acetyl-Histone H3 (Lys9) (07-352; Millipore, Billerica, MA, USA), trimethyl-Histone H3 (Lys9) (07-442; Millipore), or normal IgG (Santa Cruz Biotechnology Inc.). After cross-linking was reversed, associated plasmid DNA was collected, cleaned, and subjected to semiquantitative PCR using primer pairs flanking C/EBP-binding sites within Opn and Col11a2 promoters. The PCR products were electrophoresed on agarose gels and visualized by ethidium bromide staining. Signal intensities were measured using ImageJ software (NIH, Bethesda, MD, USA). Relative values reflecting protein–DNA interactions were calculated by adjusting corresponding signal intensities to those of input levels. PCR primers specific for pGL3-Opn and pGL3-Col11a2 reporters were developed and used to detect associated reporter plasmids in each immunoprecipitation. Primer sequences included the following: Opn F3 (forward; located at −134 to −112 from the transcriptional start site), 5′-ccacaaaaccagaggaggaagtg-3′; Col11a2 F2 (forward; located at −269 to −250 from the transcriptional start site), 5 ′-ttctgcttcacctagtccag-3 ′; and GL primer 2 (reverse; located within the pGL3 plasmid backbone), 5′-ctttatgttttggcgtcttcca-3′.
Immunoprecipitation and Western blotting
C3H10T1/2 cells transfected with the empty CMV promoter-FLAG control plasmid or FLAG-tagged EWSR1-DDIT3 expression plasmid in a 10-cm dish were lysed with 1 ml of IP Lysis Buffer (PIRCE, Rockford, IL, USA) supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail (Sigma). Immunoprecipitation of C/EBPα and C/EBPβ was performed using Dynabeads Protein A (Invitrogen Life Technologies Corp.) according to manufacturer's protocol. In brief, to 50 µl of Dynabeads Protein A, 20 µl of rabbit polyclonal anti-C/EBPα antibody (14AA, sc-61; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) or anti-C/EBPβ antibody (C-19, sc-150; Santa Cruz Biotechnology Inc.) were added and incubated for 1 h, and 500 µg protein of cell lysate added to the Dynabeads Protein A conjugated antibody for overnight on a rotating device at 4°C. Immunoprecipitated proteins were denatured for 5 min at 95°C, separated by SDS-PAGE using a 5–15% gradient gel (Bio-Rad Laboratories, Hercules, CA, USA), transblotted onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad), and subjected to Western blotting using anti-C/EBPα antibody or anti-C/EBPβ antibody (Santa Cruz). Protein bands were visualized on X-ray film using the ECL Plus Western blotting detection system (GE Healthcare, Buckinghamshire, UK).
Real-time quantitative PCR assay
After transfection of the empty CMV promoter-FLAG control plasmid or FLAG-tagged EWSR1-DDIT3 expression vector into C3H10T1/2 cells, total RNA was extracted using the SV Total RNA Isolation System (Promega, Madison, WI, USA), and 1 µg of total RNA was converted to cDNA using the GeneAmp® Gold RNA PCR Core Kit (Applied Biosystems, Foster City, CA, USA) with random hexamers as a primer in accordance with the manufacturer's instructions. For quantitative analysis of the expression levels of C/EBPα mRNA and C/EBPβ mRNA, real-time quantitative PCR (qPCR) was performed on a Real-Time PCR system (Mx3000P, Stratagene Japan K.K., Tokyo, Japan) using SYBER Premix Ex Taq™ (TaKaRa Bio, Inc. Shiga, Japan). Real-time qPCR was performed using the specific primers and levels of β-actin transcripts were used to normalize C/EBPα and C/EBPβ expression levels. Primer sequences were as follows: C/EBPα 5′-tgaacaagaacagcaacgag-3′ and 5′-tcactggtcacctccagcac-3′; C/EBPβ, 5′-gcgcgagcgcaacaacatcg-3′ and 5′-tgcttgaacaagttccgcag-3′; β-actin, 5′-tgttaccaactgggacgaca-3′ and 5′-ggggtgttgaaggtctcaaa-3′.
Each sample was analyzed in duplicate, and experiments were repeated at least three times. In all figures, data were shown as average ± standard errors (SE). All statistical analyses were performed using Microsoft Office Excel (Microsoft Corp., Redmond, WA, USA). ANOVA followed by Tukey–Kramer post-hoc test or unpaired t-test was used to determine statistical significance (p values less than 0.05 were considered significant), where applicable.