Figure 2.

Characteristics of ARN127(65) aggregation in HEK293 cells and suppression by GR derivatives. (A) Immunofluorescence of HEK293 cells cotransfected with 200 ng of ARN127 expression plasmid and 50 ng of wtGR or GRΔ108–317 expression plasmid after 48 h. Cells cotransfected with ARN127(25) and wtGR in the absence (a) or presence (b) of dex, and with GRΔ108–317 plus dex (c) all show diffuse distribution of ARN127(25). ARN127(65) cotransfected with wtGR forms cytoplasmic aggregates in the absence of dex (e), distributes diffusely in the presence of dex (f), and forms nuclear aggregates when cotransfected with GRΔ108–317 plus dex (g). A dying cell is seen with cotransfected ARN127(65) and GRΔ108–317 plus dex when viewed under immunofluorescence (d) or phase (h) microscopy. Note nuclear aggregates visible under phase microscopy. HA-tagged ARN127 is displayed in red and DAPI nuclear stain in green. (Magnifications: ×100.) (B) GR DNA binding domain and N terminus are required for suppression of polyglutamine protein aggregation. Relative suppression of ARN127(65) aggregation is shown for various GR mutants, MR, and GR-MR fusions. This valve represents the ratios of the percentage of transfected cells with aggregates minus and plus hormone administration, scored by counting >300 cells for each data point for at least three transfections. For constitutively active receptor, this value represents the ratio of the percentage of cells with aggregates in the presence (GRN525) and absence (p6R) of the receptor. Average percentage of cells with aggregates was ≈15% for wtGR − dex and ≈3.5% for wtGR + dex. Error bars represent the SEM. (C) Diagram of various GR mutants and fusions to MR. GRΔ108–317 has amino acids 108–317 deleted from its N terminus. GR(AF1–14) contains 16 amino acid substitutions within amino acids 108–317. MGG, chimera with MR N terminus replacing that of GR; GMM, chimera with GR N terminus replacing that of MR.