Pact vector

Plasmids were generated using PCR-cloning and site-directed mutagenic techniques (PCR primer sequences are provided in Supplementary Table S1). For Co-IP experiments, both full-length and truncated ELK1 coding sequences were cloned into the NotI-XhoI site of pCMV-HA-N vector (Takara Bio Inc, Japan). The full-length FOXE1 coding sequence was cloned into EcoRI-BamHI site of p3XFlag-CMV-7.1 (Sigma-Aldrich, St Louis, MO, USA). For mammalian two-hybrid experiments, the pACT-ELK1 construct was generated by cloning the ELK1 coding sequence into the BamHI-KpnI site of the pACT vector (Promega, Madison, WI, USA). The pBIND-FOXE1 construct was created by cloning the coding sequence of the FOXE1 C-terminus into the BamHI-KpnI site of the pBIND vector (Promega). For gene reporter experiments, a 474 bp region of the human TERT promoter (−391 to +83 relative to the TSS) was cloned into the KpnI-HindIII sites of the pGL3-basic vector (Promega). Generation of the pGL3-TPO reporter has been described previously [61 (link)]. Preparations of purified plasmid DNA for transfections were prepared using Qiagen’s Qiafilter plasmid maxi kit, according to the manufacturer’s instructions (Qiagen, Hilden, Germany).

pBIND vector (Promega) as a bait that includes the GAL4 DNA-binding domain and pACT vector (Promega) as a prey that includes the VP16 activation domain were used for plasmid construction. Open reading frames of TLP/mutant and p53/mutant were linked just downstream from the GAL4 DNA-binding domain of pBIND and VP16 activation domain of pACT vector, respectively. pG5-luc vector (Promega) was used as a reporter plasmid with the luciferase reporter gene.

Expression plasmid pcDNA3.1-Mef2c and 3× MEF2-luciferase reporter construct, which contain three upstream tandem repeats of a MEF2 binding site sequence from the desmin gene, were kindly provided by Eric N. Olson.
The Suv39h1 gene was amplified using SF1-SF3 primers (Table 1). Then, the recombinant plasmids were separately digested with Xho I and EcoR I or Sal I and Not I, and ligated into the pIRES2-EGFP (BD Biosciences Clontech, Franklin Lakes, NJ, USA), pCMV-Myc (BD Biosciences Clontech), and pBIND vectors (Promega, Madison, WI, USA), separately.
The Mef2c gene was amplified using MF1 primers (Table 1) designed according to the Sus scrofa Mef2c gene (accession number: NM001044540). Then, the recombinant plasmids were digested with Sal I and Not I, and ligated into the pCMV-HA (BD Biosciences Clontech), and pACT vectors (Promega), separately.
The HP1α gene was amplified using HF1-HF3 primers (Table 1). Then, the recombinant plasmids were digested with Nhe I and Xho I, and EcoR I and Xho I, or Sal I and Not I, and ligated into the pIRES2-EGFP, pCMV-HA, and pBIND vectors, respectively.