Figure 1. Wild-type p53 regulates thymine DNA glycosylase (TDG) expression. (A) siRNA targeting p53 (p53 siRNA) or TDG (TDG siRNA) or control siRNA (Scramble, SCR) was transfected in TE-1 cells which express the temperature-sensitive p53 mutant, V272M. Cells maintained at 32°C (permissive temperature) present most of the p53 in an active form, and those maintained at 37°C (restrictive temperature) present most of the p53 in an inactive form. Upper panel: detection of TDG mRNA levels by reverse-transcription quantitative PCR (RT-qPCR). Middle panel: western blotting analysis of TDG, using Ku-80 as loading control. Lower panel: detection of TDG and p53 by confocal microscopy. sip53 and siTDG: siRNA to p53 and TDG, respectively. Green fluorescence, TDG; red fluorescence, p53; blue fluorescence, nucleus (ToPro). (B) The ESCC p53-null cell line TE-13 was transfected with either 0.5, 1.0, 1.5 or 2.0 μg of DNA of an expression vector for p53 protein (p53wt-pcDNA3) or with an empty vector (pCDNA3-empty), used as control (0). Upper panel: detection of TDG mRNA levels by RT-qPCR. Lower panel: western blotting analysis of TDG, using Ku-80 as loading control. (C) TE-1 cells, cultured at either 32°C or 37°C, were treated with 0.25, 0.5, 1.0 or 2.0 mM of MMS for 3 h or not (-). Upper panel: detection of TDG mRNA levels by RT-qPCR. Middle panel: western blotting analysis of TDG, using Ku-80 as loading control. Lower panel: detection of TDG and p53 by confocal microscopy. Green fluorescence, TDG; red fluorescence, p53. (D) The isogenic breast cancer cell lines, MN1 and MDD2, were treated with 500 ng/mL doxorubicin (Dox) for 24 h andTDG mRNA levels were detected by RT-qPCR. (E) The non-transformed bronchial cells, NHBE, were treated with 0.25, 0.5 or 1.0 mM of MMS for 3 h or not (-) and TDG mRNA expression levels were assessed by RT-qPCR. Stars indicate statistical significance: *p < 0.05 (Student’s t-test using with software GraphPad Prism 4, GraphPad Software Inc.).