Fig. 5

AR coregulator switching by GV1001. A Protein–protein interaction network analysis of predicted and reported functional partners of AR. Gene interactions were constructed using the STRING online database (http://string-db.org/). Network nodes represent proteins and the edges represent protein–protein associations. B Binding probability of AR and NKX3.1 at the transcription initiation site of the human NKX3.1 and AR genes. Experimental data from the chromatin immunoprecipitation (ChIP) atlas (http://chip-atlas.org/) are shown using Integrative Genomics Viewer (IGV) software. C Expressions in normal prostate tissues, primary prostate tumor tissues, and metastatic prostate tumor tissues from prostate cancer patients (n = 164) (GSE6919). DATA were shown as box and whisker plots. Box, interquartile range (IQR); whiskers, 5–95 percentiles; and horizontal line within the box, median. D Protein interaction of YAP1 or NKX3.1 with AR. LNCaP cells were treated with GV1001 (10 μM) for 24 h, and reciprocal immunoprecipitation and immunoblotting were performed. All of the results were confirmed by multiple experiments. E GV1001 or LA-induced NKX3.1 expression. LNCaP cells were treated with GV1001 (0.1–10 μM) or LA (1–100 nM) for 24 h. F Cytoplasmic retention of YAP1 by GV1001. LNCaP cells were treated with GV1001 (10 μM) for 24 h, and subcellular fractionation and subsequent immunoblot analyses were performed. G Immunocytochemistry of YAP1 and phospho-YAP1 proteins in LNCaP cells. LNCaP cells were treated with 10 μM GV1001 for 4 h. Alexa Fluor 568 stained YAP1 (red), Alexa Fluor 488 stained phospho-YAP1 (green) and DAPI stained nuclei (blue). Magnification: 60X. H Ubiquitination induction by GV1001. LNCaP cell lysates were immunoprecipitated with YAP1 antibody and analyzed by immunoblotting with anti-ubiquitin antibody to detect polyubiquitinated YAP1. I Inhibitory effects of KT5720 (upper) and KH7 (lower) on GV1001-induced YAP1 phosphorylation. KT5720 (10 μM) or KH7 (5 μM) was added 1 h before 10 μM GV1001 treatment in LNCaP cells, and 24 h after total cell lysates were subjected to immunoblotting for phospho-YAP1 and YAP1. J mRNA levels of target genes for AR/coregulator complex. RT-qPCR was performed in LNCaP cells treated with DHT (100 nM) or GV1001 (10 μM) for 24 h. K ChIP-qPCR analysis of AR recruitment to the promoter region of PSA, CTGF or NKX3.1 gene. LNCaP cells were treated with DHT (100 nM), GV1001 (10 μM) for 24 h. Input and immunoprecipitated samples were analyzed by qPCR for PSA, CTGF, or NKX3.1 promoter region. ChIP-qPCR data are presented as a percentage of input. Data represent means ± SD (n = 6, **P < 0.01 vs vehicle-treated control)