FIGURE 4.

Colinear N-terminal fusion of SUMO restores synergy control but has no effect on AR aggregation. Human embryonic kidney 293T cells were co-transfected in 96-well plates as described under “Experimental Procedures” with expression vectors for AR forms bearing normal (Gln₂₄) or expanded (Gln₁₁₃) glutamine tracts with intact (WT) or disrupted (KRKR) SC motifs and in the absence or presence of an N-terminal fusion of SUMO3 (S3). Cells also received the constitutive pRSV β-galactosidase plasmid as well as a minimal luciferase reporter plasmid driven by either four copies of a 15-bp response element from the TAT gene (ARE-4, panel A) or a 500-bp intronic region of the AR-responsive human FKBP5 gene (panel B). Cells were treated with 10 nm R1881 or vehicle, and activity was measured as described. The data represent the average ± S.E. of at least 4 independent experiments performed in triplicate and are expressed as a percentage of the Gln₂₄ WT AR activity at each promoter, which corresponds to 25.62 ± 1.92 and 1.95 ± 0.13 for the ARE-4 and FKBP5 reporters, respectively. Expression of AR variants in 293T cells was confirmed by immunoblotting using anti-AR-N20 antibody as described under “Experimental Procedures.” C, HeLa cells expressing the indicated AR expression plasmid were treated with either vehicle or 10 nm R1881 for 20 h. The incidence of AR aggregate-positive cells was determined as in Fig. 1. Data represent the means ± S.E. of 3–4 independent experiments performed in triplicate.