Fig 2: Schematic model of XAP2 regulation of ER-dependent transcription.(A) Upon the ligand (black diamond) binding, ERa is activated, WT XAP2 is starting to be released from the promoter whereas more ERa is recruited to the promoter and the target gene transcription is initiated. (B) When XAP2 is mutated or knocked down, there is less or no functional XAP2 that could interact with ERa, ERa is recruited more actively to the target gene promoter and the transcription is highly induced.

Fig 3: XAP2 interacts with ERa but has no effect on the intracellular level of ERa.(A) HC11 cells where transiently transfected with XAP2 siRNA (msiXAP2) (lanes 2,4) or a scrambled siRNA (Scr) (lanes 1,3). 48 h after transfection, cells were treated with DMSO (-E2) or 10 nM E2 (+E2) for 1 h before harvest. Whole cell extracts were prepared and Western blot experiments were performed with indicated antibodies; ß-actin was used as a loading control. (B) The ERa protein levels shown in (A) were quantified by measuring the density of specific bands and normalizing to ß-actin progein levels. The ERa/ß-actin ratio in Scr (-E2) cells was arbitrarily set to 1 and data were expressed as means ± SE of three independent experiments. (C) HC11 cells were treated for 1 h with DMSO (-) or E2. Whole cell extract (WCE) was prepared and immunoprecipitation (IP) experiments were performed using a XAP2 antibody (lanes 3–4). The presence of ERa protein was monitored by Western blot analysis. WCE (lane 2) and an IgG antibody (lane 1) show the positive and negative controls, respectively. Data shown here is representative of three independent experiments. (D) Radioactively labeled proteins XAP2 and ERa synthesized by in vitro translation were mixed in equal amounts. After incubation (for details, see Materials and Methods), XAP2 antibodies or IgG antibodies were added to each protein mixture. Precipitated complexes were analyzed by SDS-PAGE. Data shown here is representative of three independent experiments.

Fig 4: XAP2/ERa interaction is crucial for XAP2 to inhibit ERa-mediated transcription.(A) Schematic illustration of the XAP2 protein. The locations of the PPlase-like domain and the three TPRs are showed and mutation constructs used in this study are indicated. (B) HeLa cells were transiently co-transfected with expression vectors encoding indicated XAP2 mutations together with ERa and a 3×ERE-TATA-Luc reporter. 3 h after transfection, cells were treated with DMSO or 10 nM E2 for 48 h. Whole cell extracts (WCE) were prepared and luciferase activity was measured. Reporter gene activity was determined and normalized to ß-galactosidase. Results were compared to luciferase activity of E2 dependent ERa-induced reporter activity, which were arbitrarily set to 1. (C) HeLa cells were transiently co-transfected with expression vectors encoding indicated cMyc-tagged XAP2 mutations together with ERa. 24 h after transfection, cells were treated with DMSO or 10 nM E2 for 1 h and immunoprecipitation (IP) experiments were performed using an ERa antibody. The presence of XAP2 proteins was monitored by Western blot analysis using c-Myc antibody. WCE (input) and an IgG antibody (IgG) show the positive and negative controls, respectively. XAP2 protein levels were quantified by measuring the density of specific bands. WT XAP2 transfected (+E2) cells was arbitrarily set to 1. Data were expressed as means ± SD of three independent experiments performed in triplicate. *, P<0.05 (Student's t test).

Fig 5: XAP2 represses ERa but not ERß mediated transcription.(A–B) HeLa cells were transiently co-transfected with 1 ng of ERa (A) or ERß (B) expression vectors together with increasing amounts of XAP2 (1–10 ng) together with 100 ng of a 3×ERE-TATA-Luc reporter. (C–D) HeLa cells were transiently transfected with 1 ng of ERa (C) or ERß (D) expression vectors upon increasing amounts of XAP2 (1–10 ng) together with 100 ng of a pS2 promoter luciferase reporter construct. 3 h after transfection, cells were treated with DMSO or 10 nM E2 for 48 h. Whole cell extracts (WCE) were prepared and luciferase activity was measured. Reporter gene activity was determined and normalized to ß-galactosidase. Results were compared to basic luciferase activity of the reporter constructs, which were arbitrarily set to 1. Data were expressed as means ± SD of three independent experiments performed in triplicate. *, P<0.05 (Student's t test).