Figure 1.

Ralaniten no longer inhibits the growth of LNCaP-RAL^R cells. (A) Dose response curves of LNCaP and LNCaP-RAL^R cells treated with ralaniten, bicalutamide, or enzalutamide and stimulated with 0.1 nM R1881. Data presented as mean ± SEM and analyzed by two-way ANOVA with Sidak’s test applied post hoc, n = 4 independent experiments. (B) Transcript levels of AR, PSA, FKBP5, and RHOU normalized to SDHA from LNCaP and LNCaP-RAL^R cells treated with ralaniten (35 μM), enzalutamide (5 μM), or DMSO control and stimulated with 1 nM R1881 (black bars) or EtOH vehicle (white bars). LNCaP-RAL^R cells show reduced sensitivity to ralaniten compared to LNCaP cells. Data presented as mean ± SEM and analyzed by two-way ANOVA with Dunnett’s test applied post hoc, n = 3 independent experiments. (C) Protein levels of AR and AR regulated genes from whole cell lysates treated as in panel B. (D) Western blots of AR levels in LNCaP and LNCaP-RAL^R cells transfected with control or AR siRNA (10 nM) for 24–96 h. (E) Crystal violet assay measuring proliferation of cells 96 h post-transfection with either scrambled (SCRM) or AR siRNA (10 nM) and stimulated with 0.1 nM R1881 or EtOH vehicle. Data presented as mean ± SEM and analyzed by two-way ANOVA with Dunnett’s test applied post hoc, n = 3 independent experiments. (F) Protein levels of AR and AR-variant from whole cell lysates treated as in panel B. Lysates harvested from LNCaP95 and VCaP cells serve as positive controls for AR-variant (AR-V). *p < 0.05; **p < 0.01; ***p < 0.001; ^#p < 0.0001; n.s., not significant: RAL, ralaniten; ENZ, enzalutamide.