Fig 3.
AR and NKX3-1 directly regulate each other in a feed-forward manner. AR, NKX3-1, and RNA Pol II colocalize at enhancer and promoter regions of the NKX3-1 gene. (A) Screenshots of AR, NKX3-1, and RNA Pol II ChIP-seq peaks surrounding the NKX3-1 gene. (B) ChIP-qPCR validation of AR and NKX3-1 at the enhancer and promoter of the NKX3-1 gene. DNA enrichment is represented as percentage of input chromatin immunoprecipitated. Data represent the means ± standard errors of the means (SEM) from at least three independent experiments. (C and D) Effect of AR silencing on NKX3-1 expression level. Hormone-depleted LNCaP cells were transfected with either control siRNA or siRNA targeting AR before treatment with EtOH or 10 nM DHT for 8 h. (C) Total RNA was isolated and amplified with real-time RT-qPCR primers for AR and NKX3-1. mRNA expression levels were normalized against GAPDH (glyceraldehyde-3-phosphate dehydrogenase). (D) Western blot assaying the protein levels of AR, NKX3-1, and α-tubulin after siRNA transfection. AR and NKX3-1 colocalize binding within the AR gene body. (E) Screenshots of AR and NKX3-1 ChIP-seq peaks around the AR gene. (F) ChIP-qPCR validation of AR and NKX3-1 ChIP-seq peaks within the intragenic region of AR. DNA enrichment is presented as the percentage of input chromatin immunoprecipitated. Data show the mean ± SEM from at least three independent experiments. (G and H) Effect of NKX3-1 depletion on AR expression level. Androgen-deprived LNCaP cells were transfected with either control siRNA or siRNA targeting NKX3-1 before treatment with EtOH or 10 nM DHT for 8 h. (G) Total RNA was isolated and amplified with real-time RT-qPCR primers for AR, NKX3-1, and GAPDH. mRNA expression levels were normalized against GAPDH. (H) Western analysis of AR, NKX3-1, and α-tubulin protein levels after siRNA knockdown.