Fig. 4.

FRA1 trans-activates NANOG expression upon K116 deacetylation. a FRA1 and NANOG expression analysis by western blot in empty vector (EV) and FOSL1-overexpressing DLD1 cells. b, c Relative reporter luciferase activity (b) and mRNA levels (c) of NANOG, SOX2, and LGR5 in EV and FOSL1-overexpressing DLD1 cells. d Chromatin immunoprecipitation (ChIP) analysis of DLD1 cells transfected with Flag-tagged wild-type and mutated FOSL1 expression vectors. ChIP was performed with the Flag antibody. Quantitative reverse transcriptase–polymerase chain reaction analysis was performed on immune-precipitated DNAs using a primer pair specific for the NANOG promoter. e Relative luciferase activity of wild-type and FBE-mutant (predicted FRA1-binding site) NANOG-luc reporters in DLD1 cells transfected with wild-type and mutant FOSL1 expression constructs. f DNA pull-down assay analysis of FRA1-binding ability to the biotin-labeled wild-type and FBE-mutant NANOG promoter probe (−404/−1) in DLD1 cells stably overexpressing wild-type and mutant FOSL1. Ku80 served as a control. g Relative luciferase activity of the NANOG-Luc reporter in DLD1 cells upon Tubastatin A treatment for 8 h. *p < 0.05, **p < 0.01, ***p < 0.001. Unpaired t test. Data are presented as mean ± SD