Fig. 2.

Oct4 binds to the Egr1 promoter to transactivate Egr1 in lung cancer cells. a, H1299 cells that had been cotransfected with pGL2-Egr1p and pTCY-Renilla for 24 h were transduced with LV.Oct4 or LV.Null (left panel). A549 cells stably overexpressing Oct4 and vector control cells were cotransfected with pGL2-Egr1p and pTCY-Renilla (right panel). After 48 h, cell lysates were harvested, and their firefly and Renilla luciferase activities were determined. The ratio of firefly luciferase activity to Renilla luciferase activity of vector control cells was set to 100, and the values are presented as relative promoter activities. b, Schematic representation of two deletion constructions containing different lengths of the Egr1 promoter (left panel). Numbering is relative to the translational start site at + 1. H1299 cells were cotransfected with full-length or different deletion constructs of the Egr1 promoter and pTCY-Renilla. After 24 h, the cells were transfected with pCMV-tag2B-Oct4 or the control vector pCMV-tag2B. Cell lysates were assessed for firefly and Renilla luciferase activities 48 h later. RLU, relative luciferase unit. c, ChIP analysis showing direct binding of Oct4 to the Oct4 response element (ORE)-containing region in the Egr1 promoter. Cross-linked chromatin of A549 cells was immunoprecipitated with anti-Oct4 or anti-IgG antibody combined with protein G agarose beads, followed by PCR amplification of the Egr1 promoter region encompassing the ORE (between − 309 to − 413 bp) region. Data are mean ± SEM. *, p < 0.05; ***, p < 0.001