Fig. 2

Oct4 directly binds and transactivates the M-CSF promoter in lung cancer cells. a and b Upregulation of M-CSF mRNA and protein levels in Oct4-overexpressing A549 cells (left panel) and downregulation of their levels in Oct4 knockdown A549 cells (right panel), as determined by RT-PCR (a) and ELISA (b). Expression of GAPDH served as the loading control. Values shown at the bottom of the ethidium bromide-stained gels are the ratios between the intensity of the bands corresponding to the indicated mRNA, as determined by densitometric analysis. The ratios of A549-vector cells are set as 1. c Transactivation of the M-CSF promoter by Oct4. d and e Schematic representation of a series of deletion constructs containing different lengths of the M-CSF promoter (d) and mutant constructs containing point mutations within the ORE1 and/or ORE2 sites (e). The black box indicates putative OREs. Numbering is relative to the translational start site at +1. Luciferase (Luc) reporter assays of various M-CSF promoter constructs. Two hundred ninety-three T cells were cotransfected with pSin-EF-Oct4-Pur (or control vector pSin-null) and a wild-type (c), deletion (d), or mutant (e) pFRL2-M-CSF construct. Cell lysates were harvested 48 h later, and their firefly and Renilla luciferase activities were determined using a dual-light luciferase reporter assay system. The ratio of firefly luciferase activity to Renilla luciferase activity was expressed as relative light units (RLU). f and g ChIP analysis showing direct binding of Oct4 to the ORE-containing region in the M-CSF promoter in A549-Oct (f) and H1299 (g) cells. Cross-linked chromatin was immunoprecipitated with anti-Oct4 or anti-IgG antibody combined with protein G agarose beads, followed by PCR amplification of the M-CSF promoter region that encompasses the ORE1 and ORE2 sites. Data shown represent means ± SEM (n = 3 to 4)