Fig. 7. AR 609 acetylation is required for enzalutamide-resistant CRPC tumor growth.

(A) C4–2B cells infected with a retroviral construct expressing FLAG-tagged AR or K609A-AR mutant were subjected to IP with acK609-AR antibodies, followed by immunoblotting with FLAG antibodies (top) or FLAG and actin antibodies (bottom). (B and C) Retrovirally infected C4–2B cells were grown in the presence of enzalutamide (Enz) (B) or abiraterone (Abi) (C), and cell proliferation was measured by trypan blue staining. (D) Enzalutamide-resistant C4–2B cells were infected with retrovirus-expressing AR or K609A mutant AR and injected subcutaneously in male SCID mice. Both sets of the mice were injected with enzalutamide (40 mg/kg) on days 17 and 23 after cell injection (shown by arrow). Tumor volumes were measured twice a week. (E and F) Once they reached ~1000 mm³ in volume, tumors were harvested, photographed, and weighed. Data are represented as means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, Student’s t test. (G) C4–2B and (H) VCaP xenograft tumors were treated with (R)-9b subcutaneously or orally, respectively, and were IHC-stained using pY284ACK1, pY267-AR, or acK609-AR antibodies (n = 3, per group; one representative image shown). Photographs were captured at ×20 magnification. N, nucleus; C, cytoplasm. Pathologist’s observation for intensity of the staining (range: no staining to moderate staining) is shown below the images.