(A) Gas5 suppresses mineralocorticoid (MR), progesterone-A (PR-A) and androgen (AR) receptor-induced transcriptional activity. (B) Gas5 does not influence ERα-, PPARδ-, p53- or GAL4-VP16-mediated transcription. For A and B, HCT116 cells were transfected with GR-, MR-, PR-A-, AR-, ERα-, PPARδ/RXRα-, p53-, or the VP16 activation domain (AD) fused to GAL4 DBD-expressing plasmid (GAL4-VP16AD), together with their responsive luciferase-expressing reporter plasmid and pSV40-β-Gal. Bars represent the mean +/− SEM values of luciferase activity normalized for β-galactosidase activity in the absence or presence of 10−6 M of dexamethasone (Dex) or progesterone (Prog), 10−8 M of aldosterone (Aldo), dehydrotestosterone (DHT) or estradiol (E2), 10−6 M of GW501516 (PPARδ agonist), or the indicated trans-acting factors. *: p<0.01, n.s.: not significant, compared to the baseline (in the absence of Gas5 transfection treated or transfected with indicated ligand or a trans-acting factor) (n=3). (C) Gas5 physically interacts with the DBD of the AR and PR-A, in addition to that of the GR, whereas it does not bind the ERα DBD. 10−4 M of wild type (WT) Gas5 RNA was incubated with 1 μg of GST or GST-fused DBDs of GR, MR, AR, PR-A or ERα bound to GST beads. Associated Gas5 RNA was measured with SYBR Green real-time PCR. Bars represent the mean +/− SEM values of fold association of Gas5 RNA normalized for the baseline (with GST) (n=3).