Fig. 5

KO of ABSs in the PVT1 and PCAT1 loci to assess effects on androgen-elicited MYC repression. A A schematic showing of the P11 site KO strategy. Highlighted are VCaP AR ChIP-Seq tracks, two pairs of gRNA targeting the central AR peak versus two pairs of gRNA targeting the all 5 major AR peaks, respectively. Also highlighted was a cluster of AR binding motifs enriched in the P11 site, based on motif scan analysis. B Control and P11-KO (central AR peak KO, left panel; multiple AR peaks KO, right panel) stable pools in androgen-depleted medium were subjected to 10 nM of DHT treatment for indicated time points. TaqMan RT-qPCR was conducted on MYC mRNA expression (GAPDH as internal reference), followed by data normalization with the control (vehicle) being set at 1. C Similarly, control and P4-KO stable pools in androgen-depleted medium were subjected to 10 nM of DHT treatment for indicated time points. TaqMan RT-qPCR was conducted on MYC mRNA expression (GAPDH as internal reference), followed by data normalization with the control (vehicle) being set at 1. D total RNA of the control and P4-KO pools was assessed by TaqMan RT-qPCR for the expression of the CCAT1 gene that is in proximity to the P4 site. E Total RNA of the control, P10-KO and P11-KO (central AR peak KO) pools was assessed by TaqMan RT-qPCR for expression of the PVT1 gene that is in proximity to both P10 and P11 sites. F Schematic showing of the PCAT1 SE that was in annotation with VCaP AR and H3K27ac ChIP-Seq tracks. This locus contains three lncRNAs (PCAT1, PCAT2, and PRNCR1) and several AR and H3K27ac enhancer peaks. Also highlighted was the editing strategy, aiming to excise the P1, P2, and CRE2 sites. G, H Control and 3 specific KO pools in androgen-depleted medium were subjected to 10 nM of DHT treatment for indicated time points. TaqMan RT-qPCR was conducted on MYC mRNA expression (GAPDH as internal reference), followed by data normalization with the control (vehicle) being set at 1. Data are mean values ± SD for three biologically independent samples. P values are two-sided Student’s t test. *P < 0.05; **P < 0.01. N.S., no statistical significance. See Additional file 1: Figs. S6, S7 for additional information