Figure 3

NKX3-1 mediates androgen-regulation of TWIST1. A. Three putative NKX3-1-bindingsites (BS1, BS2 and BS3) are shown as boxes in the TWIST1 promoter (from NCBI MapViewer). B. Chromatin immunoprecipitation was performed using chromatin isolated from LNCaP cells stimulate with 10 nM R1881 (+R1881) for 24 hours or left untreated (−R1881) and using a monoclonal antibody against NKX3-1 (NKX3-1). sqPCR results using primers targeting BS3 are shown in B. The results of the BS3 primer set are shown adjusted to the levels of chromatin immunoprecipitated with IgG as a negative control. Data are presented as mean ± SD (n=3). C. LNCaP cells were transfected with pGL3-TWIST-Luc (pGL3-TWIST) and a human NKX3-1 expression plasmid or an empty vector (pCR3) as well as pCMV β-gal. As control, LNCaP cells were transfected with a reporter plasmid lacking the TWIST promoter region (pGL3) and the human NKX3-1 expression vector or an empty expression plasmid (pCR3). The luciferase activities were normalized against the corresponding β-galactosidase activities and the results are shown as mean ± SD (n=3). A paired t-test was performed and a two-tailed p value <0,05 is indicated with a * D. LNCaP cells were transfected with 100 nM of siRNA targeting NKX3-1 or a non-silencing siRNA (ctr.) and total RNA was harvested 72 hours after transfection. TWIST1 mRNA expression was examined using sqRT-PCR. The data are shown as mean ± SD (n=3) relative to the levels of LNCaP cells transfected with a non-silencing control siRNA. E. Representative Western blots showing expression levels of NKX3-1 in (from top) LNCaP cells stimulated with 10 nM of R1881, transfected with NKX3-1 overexpression vector or targeted by siRNA against NKX3-1. α- tubulin is shown as loading control.