Fig. 4

CRISPR/Cas9-mediated genomic knock-out (KO) of P10 to evaluate its impact on androgen-elicited MYC transrepression. A A schematic showing of the MYC gene locus with the P10-KO strategy. Highlighted are VCaP AR ChIP-Seq tracks, two pairs of P10 targeting gRNA (U2/U1 for upstream gRNAs under G418 selection; and D1/D2 for downstream gRNAs under Puro selection), together with PCR and DNA-Seq primers. B Genomic DNA of stable pools were amplified by round-1 PCR. The predicted PCR product sizes: control pool (995 bp) and P10-KO pools (371 bp, 317 bp, 313 bp, and 259 bp). C The round-1 PCR products were re-amplified by round-2 PCR (nest-PCR). The predicted PCR product sizes: control pool (941 bp), P10-KO pools (316 bp, 262 bp, 258 bp, and 204 bp). D as loading controls, control PCR tests were conducted with indicated primers to amplify the MYC and PSA loci that are not targeted by gRNA. Arrow heads indicated bands with expected sizes. E The identities of the major nest-PCR bands were assessed and verified by band excision, DNA purification, DNA-Seq, and alignment. The results indicated that editing predominantly occurred at U1 and D2 gRNA sites. F Control (C) and P10-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). The normalized read-outs were shown, with the vehicle-treated control samples 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. See Additional file 1: Fig. S4, S5 for additional information