Fig 1: AP-2a inhibits Nanog expression. (A) qRT-PCR was performed to elucidate the effect of AP-2a on the expression of stem cell markers. The relative expression of each gene in the U251 AP-2a and A172 AP-2a groups was normalized to that in the U251 NC and A172 NC groups, which were set to 1 as the control. (B) Western blots were performed to detect the expression of stem cell markers in AP-2a-overexpressing U251 cells (left) and subcutaneous mouse tumor tissues from U87 cells (right). (C, D) Luciferase assays were performed to detect the effects of AP-2a on the transcriptional activities of Nanog regulatory regions, including wild-type and mutated AP-2 binding sites. (C) Luciferase activity was normalized to ß-galactosidase activity and the results were presented as the mean ± SD of three independent transfection experiments in triplicate. (D) Data are presented as the fold change relative to Nanog wild-type reporter activity inhibited by AP-2a, which is considered 100%. (E) Electrophoretic gel mobility shift assays of AP-2a direct binding to the Nanog regulatory region in vitro. GST-AP-2a fusion protein was incubated with biotin-labeled AP-2 binding oligonucleotides from Nanog regulatory region. Shift band indicates protein-DNA complexes. (F) Immunofluorescent staining confirmed the decreased expression of Nanog in AP-2a-overexpressing U251 cells. Relative fluorescence intensity was quantified using ImageJ software. (G) The correlation of AP-2a and Nanog expression in glioma tissues was analyzed using GraphPad Prism. WT, wild-type. *, p<0.05, **, p<0.01, compared with controls.