Fig 1: MTDH binding to the RKIP promoter. (A) Chromatin immunoprecipitation (ChIP) assay. MCF-7 cells were double cross-linked with DSG and 4% formaldehyde and lysed with sonication. DNA fragments were immunoprecipitated with anti-MTDH antibodies (MTDH) or IgG as a negative control (IgG). Specific DNA fragments were amplified with a pair of primers. For input control, either none or purified DNA fragments were amplified. PCR primers used for ChIP assay were created to specifically amplify a 256 bp-DNA fragment (from -83 to +168 on the upstream region of RKIP gene, as shown in the top of (A). Amplified DNA fragments were separated on a 1% agarose gel and visualized by EtBr staining. The first lane indicates 100 bp size markers. (B) A schematic illustration of the constructs for the luciferase RKIP promoter assay. The serially deleted RKIP promoter DNA fragments (-806/+168, -428/+168, -83/+169, -48/+168, and +/+168) were inserted into the pGL4.20 luciferase vector. (C) The RKIP promoter activity. The relative luciferase activities were determined in MCF-7 cells transfected with serially deleted constructs of the RKIP promoter. Data represent fold-changes from three independent experiments. ** <0.01, * <0.05 p-value. (D) RKIP promoter activity upon modulating MTDH expression. MCF-7 cells were transfected by none (ctr), shMTDH (kdMTDH), FLAG-MTDH (oeMTDH), or VEZF1 plasmids (oeVEZF1) and used in luciferase assay with the RKIP promoter -83/+168 construct. Data represent fold changes from five independent experiments. ** <0.01, * <0.05 p-value.