Figure 3.

(A-B) LNCaP cells treated with AZD3514 for 24 hours. Graph shows AR levels normalized to GAPDH and relative to the vehicle control. Data is represented as Mean of 3 independent experiments. Error bars are SEM.

(C) LNCaP cells grown in steroid free conditions were incubated with 10 μM AZD3514, enazalutamide or vehicle control for the indicated times. Cells were then treated −/+ 1 nM DHT for 30 minutes. Effects on total AR were assessed by Western blot and normalized to levels of GAPDH. * indicates reduced AR expression.

(D) LNCaP cells grown in steroid free conditions were treated with 1 or 10 μM of AZD3514 or enzalutamide for 2 hours and then −/+ 1 nM of DHT for 30 minutes. Levels of cytoplasmic and nuclear AR were assessed by Western blotting. Levels of PARP and GAPDH were used to assess nuclear and cytoplasmic fractionation.

(E-G) AR U2OS cells, Recombinant U2OS cells stably expressing human androgen receptor (AR) fused to the C-terminus of enhanced green fluorescent protein, were dosed with AZD3514 or enzalutamide for 30 minutes before treatment of cells with 0.3 nM DHT for 2 hours prior to fixation. Representive images at 20x magnification include (i) Vehicle (ii) DHT (iii) 10 μM AZD3514 + DHT (iv) 10 μM enzalutamide + DHT. (F-G) Inhibition of DHT induced AR foci formation and AR translocation as measured on Arrayscan VTI using a compartmental analysis bioapplication. Data are representative from one experiment. Error bars are SD.