Over-expression of p53 and TFAM by plasmid transfection

The open reading frame (ORF) of p53 in pEnter (p53-pEnter), ORF of TFAM in pEnter (TFAM-pEnter) and empty vector plasmid pEnter (Empty-pEnter, Transheep Bio, Shanghai, China) were transfected into HCT116 cells by using LipoFiter (Shanghai Hanheng, Shanghai, China) according to the manufacturer's protocol. In short, HCT116 cells which were in logarithmic growth phase were seeded on 6 cm dishes with the density of 5×10⁵ cells per dish. p53-pEnter, TFAM-pEnter or empty-pEnter DNA (3 μg) and LipoFilter (20 μL) were added to each well when HCT116 cells grew to around 50-70% confluence. Then, after 6 h of transfection, serum-free medium was replaced with fresh PRMI-1640 and cultured for another 72 h after which subsequent experiments were performed. p53 and TFAM protein overexpression were both verified by qRT-PCR and Western blot.