Figure 2

Regulation of NRP1 expression by the androgen signaling axis. (a) Relative NRP1 mRNA expression in LNCaP cells grown in CSS after 48-h treatment with 10 nM DHT, 1 nM R1881 or vehicle. (b) Modified UCSC screenshot showing AR binding sites (ChIP-seq) proximal to the NRP1 gene in 13 PCa samples ²² (GSE70079). Each track depicts ChIP-seq AR binding intensity for a given sample. (c) ChIP-quantitative PCR (qPCR) demonstrates AR binding to distal 1 and Intron_12 regions at the NRP1 gene locus. The dotted line demarcates no enrichment over an IgG control ChIP. A known AR binding site in the KLK3 enhancer region was used as a positive control, whereas a gene-poor region on chromosome 20 with no previous evidence of AR binding was used as a negative control (NC). (d) NRP1 expression in LNCaP cells grown in CSS after 48- h treatment with 10 nM DHT and AR or scrambled control (scr) small interfering RNA. For AR and KLK3/PSA mRNA levels refer to Supplementary Figure 1. (e) NRP1 expression in LNCaP cells grown in CSS after 48- h treatment with 10 nM DHT with or without Bic or ENZ co-treatment. (f) qPCR analysis of NRP1 and PSA expression and (g) western blot analysis of NRP1 expression in LNCaP cells after culture in CSS for 1, 3, 5 or 7 days, or 7 days followed by 3 days of DHT treatment (10 nM). (h) NRP1 mRNA expression levels in LuCaP35 xenografts following sham castration (sham) or castration (Cx) of host mice. Raw expression data from GSE33316.⁵¹ *P<0.05; **P<0.01; ****P<0.0001.