Laschak et al. BMC Cancer 2012, 12:130 http://www.biomedcentral.com/1471-2407/12/130 Page 6 of 10 Figure 4 NO does not influence hormone-induced nuclear translocation of the AR. Prostate cancer cells (PC-3) expressing a green fluorescent AR-EosFP [37] were incubated for 6 hours in the absence/presence of DHT and JS-K/JS-Kneg. Intracellular localization of AR-EosFP was determined by fluorescence microscopy: (A) untreated controls, (B) 5 nM DHT, (C) 5 nM DHT and 4 μM JS-K, (D) 5 nM DHT and 4 μM JS- Kneg. Bars: Data presented in % cellular localization ± SD. Results are mean values of three independent experiments performed in quadruplicates. means of a colorimetric MTT assay, measuring the reduc- tion of tetrazolium salts to formazan derivatives by func- tional mitochondria [32,33]. Although redox sensitive viability assays have some limitations depending on the chemical nature of the compounds to be evaluated [45] they are widely used for measuring cell viability following nitrosative or oxidative stress [22,46,47]. The suitability of this method was furthermore documented by a previous study analyzing the effects of the spontaneous NO-donor DETA/NO on the viability of PCa-cells. Comparison of MTT with three commonly used methods to measure cell proliferation, i.e quantification of intracellular ATP, trypan blue dye exclusion and neutral red assay were found to generate similar results [22]. A first series of experiments indicated that castration resistant 22Rv1 cells were more susceptible to the growth inhibitory effects of JS-K than the androgen sensitive LNCaP cells (Figure 7A). Maximal growth inhibitory effects were achieved at a concentration of 4 μM JS-K for 22Rv1 (growth inhibition 50 ± 6%, p < 0.05) and 4 μM JS-K for LNCaP (growth inhibition 23 ± 9%). Given the highly divergent nature of the AR-positive cell lines 22Rv1 and LNCaP we repeated the proliferation experiments using the androgen sensitive LNCaP and LNCaP-SSR, an AR-positive castration resistant LNCaP- subline. The AR-negative DU-145 served as control (AR Figure 5 Influence of JS-K and JS-Kneg on the androgen receptor concentration in human prostate cancer cells. 22Rv1 and LNCaP cells were incubated for 30 hours with or without 5 nM dihydrotestosterone in the presence of JS-K or JS-Kneg. Subsequently cells were lysed and separated by SDS-electrophoresis (15 μg protein per lane). Proteins were transferred onto a nitrocellulose membrane. AR and b-actin (loading control) were visualized by immunodetection. Figure 6 JS-K diminishes WNT-signalling in 22Rv1 cells. 22Rv1 cells were co-transfected with an expression vector for mutated, stabilized b-catenin together with either the TCF reporter construct TOP or FOP as recently described [25]. Data are presented as fold of untreated controls (TOP/FOP) ± SD, **p < 0.01, ***p < 0.001.