PMC

A) XTT assay of PC12-AR112Q cells treated with R1881 (48 hours) together with either B2 or vehicle (72 hours) shows that cell survival is decreased by ligand, and that this effect is attenuated by B2. Graph, mean ± s.e.m., n = 3, * p = 0.05, (post-hoc t-test).
B) Caspase 3 assay of MN-1 cells stably expressing AR65Q and treated as indicated for 48 hours shows that B2 decreases caspase 3 activity, but has no effect on the caspase 3 activation induced by staurosporin (inset). The caspase inhibitor z-VAD-FMK (10 µM, 48 hours treatment) and the caspase activator staurosporin (1 µM, 6 hours treatment) were used as controls. Graph, mean ± s.e.m., n = 3, * p = 0.004, (post-hoc t-test).

(Upper panel) Transgenic flies expressing AR52Q in the eye were fed with dihydrotestosterone (DHT) and either vehicle or B2. Exposure of the flies to DHT resulted in the alteration of the eye phenotype, which was attenuated by B2. A magnification of the posterior side of the eye is shown on the right of each panel. (Middle panel) Quantification of disease severity is shown in the graph (see “Material and Methods” section). Graph, mean ± s.e.m., n = 45 for the DHT-fed flies and 57 for the DHT/B2-fed flies, * p = 0.001 (two-sample t-test). (Bottom panel) Western blotting analysis of AR transgene expression levels reveals that B2 does not change mutant AR expression in flies. Tubulin (Tub) is shown as loading control.

A and B) Transcriptional assay of HEK293T cells transfected with vectors expressing AR65Q (A) or as indicated (B) and the reporter vectors pARE-E1b-luc and pCMVβ for luciferase and beta-galactosidase expression, respectively, and treated with B2 (48 hours) and R1881 (24 hours) shows that B2 reduces mutant AR transactivation. Data are represented relative to AR65Q-expressing cells treated with 10 nM R1881, which are set as 100%. Graphs, mean ± s.e.m., n = 3 independent experiments, (A) * p = 0.05 and ** p = 0.001, (B) R1881 10 nM, * p = 0.004, (post-hoc t-test).
C) Transcriptional assay of HEK293T cells transfected with the pFHRE-luc and pCMVβ reporter vectors, treated with B2 (48 hours) and either serum-starved or treated with IGF-1 for 24 hours revealed that B2 does not affect pFHRE reporter activity. Data were analyzed as described in (A). Graph, mean ± s.e.m., n = 3.
D) Transcriptional assay of HEK293T cells transfected with the AR expression vectors indicated and the reporter vectors as in (A) shows that B2 is active on the acetylation-defective AR mutant. Data were analyzed as in (A). Graph, mean ± s.e.m., n = 3, * p = 0.02 (post-hoc t-test).
E) Ligand binding assay of HEK293T cells transfected with vector expressing AR65Q, treated with radioactive ligand for two hours, then treated with either B2 or cold ligand for 1 hour, shows that B2 does not compete with ligand for binding to mutant AR. Schatchard analysis shows that B2 does not compete for binding with radioactive ligand. Graph, mean ± s.e.m., n = 3 independent experiments.
F) Ligand binding assay of HEK293T cells transiently expressing normal AR and treated with either vehicle (AR24Q) or 10 µM B2 (AR24Q + B2) shows that B2 does not alter binding of normal AR to ligand. Graph, mean ± s.e.m., n = 3 independent experiments.

A) PC12 cells stably expressing mutant AR (AR112Q) were induced with doxycycline, treated with vehicle, B2 (72 hours) and R1881 (48 hours) as indicated and processed for immunocytochemistry. AR was detected with N20 antibody (green) and nuclei with DAPI (blue). Quantification of the number of cells with AR-positive nuclear inclusions and of cells with nuclear diffused AR is shown at the bottom. Graph, mean ± s.e.m., n = 4, * p = 0.002 (post-hoc t-test). Bar, 10 µm.
B) (Upper panel) Western blotting of PC12-AR112Q cells showing AR protein in cells treated with R1881 and B2 as indicated in (A). Actin is shown as loading control. Shown is one experiment representative of three. MW, molecular weight. (Bottom panel) Quantification of mutant AR aggregation reveals that B2 increases AR aggregation in the absence of ligand, but has no effect on aggregation in the presence of ligand. HMW, high molecular weight. Graph, mean ± s.e.m., n = 3, * p = 0.02, NS non-significant (post-hoc t-test).
C) Nuclear-cytoplasmic fractionation of HEK293T cells transiently transfected with vector expressing AR65Q and treated as indicated shows that B2 does not affect nuclear translocation induced by ligand. Shown is one experiment representative of three. N, nuclear fraction; C, cytosolic fraction.
D) Western blotting analysis of PC12-AR112Q cells treated as described in (A) shows that B2 treatment does not change the expression levels of Hsp90, Hsp70, and Hsp40. Actin is shown as loading control. This is one experiment representative of three.