Fig. 1. AR Lys⁶⁰⁹ acetylation is enhanced in enzalutamide- and abiraterone-resistant CRPC.

(A) Resistant C4–2B cells were treated with 7 μM enzalutamide or abiraterone for 7 days, and the cell viability was measured via a trypan blue exclusion assay (n = 3, three replicates). (B) Enzalutamide-resistant C4–2B and LNCaP cells were subjected to IP using acK609-AR antibody, followed by immunoblotting with AR antibodies (top). The same lysates were also subjected to immunoblotting with AR and actin antibodies (bottom). (C to E) Modulation of acK609-AR with (C) SBHA (1 μM, 24 hours), (D) C646 (5 μM, 24 hours), and (E) ACK1 inhibitor (R)-9b (5 μM, 24 hours) treatment in VCaP, LNCaP, and C4–2B prostate cancer cells. Endogenous acK609-AR was detected by IP using ac609-AR antibody, followed by immunoblotting with AR antibodies (top). Relative expression of acK609-AR and AR is shown. For (B to E), shown below each blot is the densitometric measurement of change in abundance relative to control. (F) Venn diagrams (VDs) summarizing the overlap between sites bound by acK609-AR and AR in VCaP cells. (G) VDs summarizing the overlap between sites bound by acK609-AR in enzalutamide- and vehicle-treated LNCaP cells. (H to K) VCaP cells were treated overnight with vehicle, (R)-9b (5 μM), SBHA (1 μM), or C646 (5 μM), and chromatin was immunoprecipitated (ChIP) with ac609-AR antibody, followed by qPCR with primers corresponding to (H) AR enhancer AREM1, (I) ACK1 enhancer, (J) PDCD6IP intron, or (K) NRG3 intron. For (H to K), n = 2, three replicates; representative data are shown. Data are represented as means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, Student’s t test. DMSO, dimethyl sulfoxide; IgG, immunoglobulin G.