Figure 6.

TTTY15 interacts with DNA methyltransferases (DNMT) 3A and decreases the binding of DNMT3A to the TBX4 promoter. (A) Cell fractionation assays were performed to determine the distribution of TTTY15 expression between the cell cytoplasm and nucleus. (B) TTTY15 RNA coimmunoprecipitated with DNMT1, DNMT3A, or IgG was quantified by qRT-PCR. PCR products were then loaded onto a 3% agarose gel for confirmation. (C) DNMT3A turned out to be upregulated in human NSCLC samples compared to the adjacent normal tissue samples (n = 37 for each group). (D) The knockdown of DNMT3A increased the expression levels of TBX4 in A549 and H441 cells. (E) The knockdown of TTTY15 did not affect the protein expression levels of DNMT3A. (F) Chromatin immunoprecipitation (ChIP)-qPCR was performed to quantify the binding of DNMT3A to the TBX4 promoter (P1: −1734 to −1626 bp; P2: −740 to −672 bp). (G) Methylation-specific PCR was performed to analyze the methylation status on the CpG islands of the TBX4 promoter in A549/scramble and A549/shTTTY15 (Me: methylated; Un: unmethylated; H₂O was used as a negative control). Data are presented as the mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001. Statistical analysis was conducted by Student’s t-test (n = 3).