Fig. 3: AR directly blocks the expression of NK1R in AdPC cells.

A Protein level of NK1R in 22Rv1 and LNCaP cells treated with ENZ and/or CSF for 1 week. B CHIP-Seq data (GSE14097) in hormone-starved LNCaP cells treated with ethanol or R1881 is re-analyzed and ChIP-SEQ signal around NK1R gene is indicated (ARE1–4). C Protein level of NK1R in 22Rv1 and LNCaP cells treated with DHT (0–1 nM) for 36 h. D ChIP of AR antibody was performed in 22Rv1 and LNCaP cells which are pre-incubated in a medium with 10% CSF for 1 week and then treated with 1 nM DHT for 36 h; QPCR was performed with primers flanking NK1R gene promoter or enhancer regions (ARE1–4). IgG is used as a negative control antibody. E ChIP-QPCR of AR antibody was performed in 22Rv1-NE and LNCaP-NE cells with primers flanking the potential ARE sites of NK1R gene expression regulatory regions (ARE1–4) as described. F Genomic DNA PCR for CRISPR-Cas9 mediated ARE1 deletion in 22Rv1 and LNCaP. Cells were transfected with plasmids of Lenti-sgARE1 or control Lenti-CRISPR for 48 h and harvested for genomic DNA PCR. ACTIN was used as a loading control. G ChIP-QPCR of AR antibody was performed in 22Rv1 and LNCaP cells transfected with plasmids of Lenti-sgARE1 or control Lenti-CRISPR with primers flanking the ARE1 region as described. H Protein level of NK1R in 22Rv1 and LNCaP cells transfected with plasmids of Lenti-sgARE1 or control Lenti-CRISPR. Cells were treated with or without ENZ and/or CSF for 1 week and the whole cell lysates were analyzed by Western blot. I Expression of NK1R and NE signature genes in 22Rv1 and LNCaP cells transfected with plasmids of Lenti-sgARE1 or control Lenti-CRISPR. Data were shown as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001.