Journal: Journal of Biological Chemistry

Article Title: Repressors of Androgen and Progesterone Receptor Action

doi: 10.1074/jbc.m305153200

Figure Lengend Snippet: FIG. 1. SMRT, DAX-1 and NCoR repress AR activity. A, HeLa cells were transfected with 5 ng of pCR3.1 AR, 400 ng of GRE2 E1b Luc reporter, 30 ng pCR3.1 -Gal, and either 150 ng of pCR3.1 vector or increasing concentrations of pCR 3.1 SMRT or DAX-1 supplemented with the pCR3.1 vector to a total amount of 150 ng. After transfection, cells were either left untreated (open bars) or treated with 3 nM R1881 (filled bars). 24 h later cells were harvested and assayed for luciferase and -galactosidase activity. Relative luciferase units (RLU) were normalized for -galactosidase activity. Each data point was done in triplicate, and the average and S.D. calculated. B, HeLa cells were transfected and assayed as in A, except after transfection they were either left untreated (open bars) or treated with 100 nM RU486 (gray bars). C, HeLa cells were transfected with 8 ng of AR and identical amounts of reporter, pCR3.1 -galactosidase, and the indicated amounts of NCoR balanced with the pSG5 vector to 200 ng. Cells were treated (no treatment, open bars; 3 nM R1881, filled bars, 100 nM RU486, gray bars) and assayed as in A. D, COS-1 cells were transfected as in C. After transfection, cells were either left untreated (open bars), treated with 3 nM R1881 (filled bars) or treated with 100 nM RU486 (gray bars). E, 40 g of cellular extracts from the transrepression experiment in A and B were resolved on SDS-PAGE, transferred to a nitrocellulose membrane and AR detected as described under “Experimental Procedures.”

Article Snippet: Trichostatin A was obtained from Sigma Aldrich, mifepristone (RU486) from Siniwest Holdings (San Diego, CA), Methyltrienolone (R1881) and Promegestone (R5020) from PerkinElmer Life Sciences (Boston, MA).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Luciferase, SDS Page, Membrane

Journal: Journal of Biological Chemistry

Article Title: Repressors of Androgen and Progesterone Receptor Action

doi: 10.1074/jbc.m305153200

Figure Lengend Snippet: FIG. 4. Analysis of physical interaction of AR and DAX-1 and PR with SMRT and DAX-1. A, protein extract (4 mg) prepared from 3 nM R1881-treated LNCaP cells was immunoprecipitated with either DAX-1 rabbit polyclonal antibody or nonspecific rabbit anti-mouse IgG (RAM) antibody. Immunoprecipitated proteins were resolved on SDS-PAGE and analyzed by Western blotting with AR 441 antibody. I, 10 g of LNCaP protein extract. B, 1 g of polyhistidine-tagged PRB and 50 l of [35S]methionine labeled in vitro translated DAX-1, were coimmunoprecipitated with either DAX-1 or RAM antibody. Precipitated proteins were eluted and run on an SDS-PAGE gel along with the input. PR was detected by Western blotting with 1294 PR antibody. I, 100 ng of PR; C, 50 l [35S]methionine labeled DAX-1 was coimmunoprecipitated with PR6 antibody in the presence or absence of 2 g of polyhistidine-tagged PRB. The eluted proteins were run on an SDS-PAGE along with 2 l of starting 35S-labeled DAX-1 in vitro translation mix (I), transferred to nitrocellulose, and the membrane was exposed to X-OMAT AR film for 30 min to detect 35S-labeled DAX-1. D, 3 mg of cleared protein extract from T47D cells treated with 100 nM R5020 were used in an immunoprecipitation with either DAX-1 or RAM antibody. Proteins were extracted as described in methods, run on a 6.5% SDS-PAGE gel and PR detected by Western blot using the 1294 antibody. I, 5 g T47D extract. E, 2 mg of precleared T47D cell extract prepared from cells treated with either 100 nM R5020 (left panel) or 10 nM RU486 (right panel) were used for immunoprecipitation with either SMRT and RAM antibody or RAM antibody only. On the left of each gel are 5 and 1 g of protein extracts from T47D cells treated with the indicated ligand. On the right are the immunoprecipitated proteins. Precipitated PR was detected by Western blot analysis using 1294 PR antibody.

Article Snippet: Trichostatin A was obtained from Sigma Aldrich, mifepristone (RU486) from Siniwest Holdings (San Diego, CA), Methyltrienolone (R1881) and Promegestone (R5020) from PerkinElmer Life Sciences (Boston, MA).

Techniques: Immunoprecipitation, SDS Page, Western Blot, Labeling, In Vitro, Membrane

Journal: Journal of Biological Chemistry

Article Title: Repressors of Androgen and Progesterone Receptor Action

doi: 10.1074/jbc.m305153200

Figure Lengend Snippet: FIG. 5. TSA does not relieve repression of AR by SMRT and coactivators relieve AR repression by DAX-1. A, HeLa cells were transfected with 5 ng of pCR3.1 AR, 30 ng of pCR3.1 -galactosidase, 400 ng of GRE2 E1b Luc reporter, and either pCR3.1 vector or increasing concentrations of pCR3.1 SMRT. After transfection cells were either left untreated (open bars), treated with 3 nM R1881 (filled bars), or 3 nM R1881 and 400 nM Trichostatin A (TSA) (gray bars). B, cells were transfected with 5 ng of pCR3.1 AR, GRE2 E1b Luc reporter, 30 ng pCR3.1 -Gal, 65 ng of pCR3.1 or pSG5 or DAX-1, and increasing concentrations of SRC-1 balanced with pCR3.1 to a total of 300 ng, or increasing concentrations of ARA70 balanced with pSG5 to a total of 300 ng of DNA. Cells were either left untreated or treated with 3 nM R1881. Cellular extracts were assayed for luciferase and -galactosidase activity, and RLU values were normalized for -galactosidase activity. Filled bars, R1881-treated samples balanced with pCR3.1 for analysis of SRC-1 activity.

Article Snippet: Trichostatin A was obtained from Sigma Aldrich, mifepristone (RU486) from Siniwest Holdings (San Diego, CA), Methyltrienolone (R1881) and Promegestone (R5020) from PerkinElmer Life Sciences (Boston, MA).

Techniques: Transfection, Plasmid Preparation, Luciferase, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Repressors of Androgen and Progesterone Receptor Action

doi: 10.1074/jbc.m305153200

Figure Lengend Snippet: FIG. 6. The SRC-1 coactivator interacting site differs from the DAX-1 interacting site in AR. A, 400 ng of 17-mer Luc and the indicated combinations of either 500 ng of Act or Act ARABC, 100 ng of Bind or Bind SRC-1, and increasing concentrations of DAX-1 balanced with pCR3.1 to a total of 100 ng were transfected; 24 h post-transfection cells were assayed for luciferase activity. RLU values were normalized to total protein concentration. B, cells were transfected as in A except Bind ARDH and Act SRC-1 were used and cells were either treated with 10 nM R1881 (filled bars) or left untreated (empty bars). C, cells were transfected as in A except Act AR and Bind SRC-1 were used. After transfection cells were either left untreated (empty bars) or treated with 10 nM R1881. 24 h later cells were harvested and assayed for luciferase activity that was normalized for protein concentration. D, structure of the constructs used in the interaction assays as previously reported (11). Full-length AR was fused to Act (an activation domain of VP16). Act ARABC contains the N terminus, DNA binding domain and hinge regions (dotted area) fused to the VP16 activation domain (Act). Bind ARDH contains the DNA binding domain, hinge, and LBD of AR fused to Bind (DNA4 DBD). Act PR contains the VP16 activation domain fused to full-length PRB. Bind PRLBD chimera has the GAL4 DBD fused to the hinge and LBD of PR. Bind SRC-1a is a full-length SRC-1a with the GAL4 DBD fused to the N terminus.

Article Snippet: Trichostatin A was obtained from Sigma Aldrich, mifepristone (RU486) from Siniwest Holdings (San Diego, CA), Methyltrienolone (R1881) and Promegestone (R5020) from PerkinElmer Life Sciences (Boston, MA).

Techniques: Transfection, Luciferase, Activity Assay, Protein Concentration, Construct, Activation Assay, Binding Assay

Journal: Journal of Biological Chemistry

Article Title: Repressors of Androgen and Progesterone Receptor Action

doi: 10.1074/jbc.m305153200

Figure Lengend Snippet: FIG. 7. Effect of corepressors on protein-protein interactions of AR and PR. A, HeLa cells were transfected with 400 ng of 17-mer Luc reporter and the indicated combinations of either 100 ng of Bind or 100 ng of Bind ARDH, 500 ng of Act, or 500 ng of Act ARABC, and increasing amounts of DAX-1 balanced with pSG5 vector to a total of 500 ng. Cells were either left untreated (open bars) or treated with 10 nM R1881 (filled bars). B, cells were transfected as in A and increasing amounts of SMRT balanced with pCR3.1 to a total of 500 ng were added instead of DAX-1. Cells were left untreated (open bars) or treated with 10 nM R1881 (filled bars). C, HeLa cells were transfected with 17-mer Luc reporter, and the indicated combinations of 20 ng of Bind or Bind SRC-1, 100 ng of Act, or Act PR, and increasing concentrations of DAX-1 balanced with the vector. After transfection, cells were either treated with 100 nM R5020 (filled bars) or left untreated (open bars). Cells were lysed and assayed for luciferase activity. RLU values were normalized to protein concentration. D, cells were transfected as in C except 20 ng of Bind PRLBD was used. Reporter activity was normalized for protein levels.

Article Snippet: Trichostatin A was obtained from Sigma Aldrich, mifepristone (RU486) from Siniwest Holdings (San Diego, CA), Methyltrienolone (R1881) and Promegestone (R5020) from PerkinElmer Life Sciences (Boston, MA).

Techniques: Protein-Protein interactions, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Protein Concentration

Journal: Journal of Biological Chemistry

Article Title: Repressors of Androgen and Progesterone Receptor Action

doi: 10.1074/jbc.m305153200

Figure Lengend Snippet: FIG. 8. Domains of AR, PR, and DAX-1 necessary for functional interaction. A, HeLa cells were transfected with 400 ng of GRE2 E1b Luc reporter, 40 ng of pCR3.1 -Gal, 5 ng of AR LBD (amino acids 1–660), and either increasing concentrations of DAX-1 balanced with its expression plasmid pSG5 or increasing concentrations of SMRT balanced with the corresponding expression vector pCR3.1. Luciferase activity was normalized for -galactosidase expression. B, cells were transfected with 400 ng of GRE2 E1b Luc reporter, 40 ng of pCR3.1 -Gal, and the indicated amounts of PR and repressors balanced with the corresponding vector to a total of 40 ng. Cells were either left untreated (open bars) or treated with the appropriate agonist (10 nM R5020 for PR and 3 nM R1881 for AR) (filled bars). Ratios of 1 to 2.5 and 1 to 8 were found to be optimal for PR repression by DAX-1 and SMRT, respectively, and the repression by these levels is shown here. C, 400 ng of GRE2 E1b Luc reporter, 40 ng of pCR3.1 -Gal, and increasing concentrations of PRLBD were transfected into HeLa cells in the presence of pSG5 vector to demonstrate the receptor dependence of the luciferase activity. The highest level of receptor mutant (60 ng) was also transfected with increasing concentrations of pSG5 DAX-1 balanced with vector to test the effects of DAX-1 on activity. Luciferase activity was normalized for -galactosidase activity. D, cells were transfected as in C except pCR3.1 SMRT balanced with pCR3.1 vector to 500 ng was used instead of DAX-1. E, cells were transfected with 400 ng of GRE2 E1b Luc reporter, 40 ng of pCR3.1 -Gal, 5 ng of AR, and increasing concentrations of either pCR3.1 DAX-1, or pCR3.1 DAX-11–226, or pCR3.1 DAX-1220–413, all balanced with the pCR3.1 vector to the same amount of DNA. Cells were either treated with 3 nM R1881 (filled bars) or not (open bars) for 20 h, harvested, and assayed for luciferase and -galactosidase activity. F, cells were transfected as in E, except 5 ng of PR was used. Cells were left untreated (open bars) or treated with 10 nM R5020 (filled bars) for 20 h, and assayed for luciferase and -galactosidase activity.

Article Snippet: Trichostatin A was obtained from Sigma Aldrich, mifepristone (RU486) from Siniwest Holdings (San Diego, CA), Methyltrienolone (R1881) and Promegestone (R5020) from PerkinElmer Life Sciences (Boston, MA).

Techniques: Functional Assay, Transfection, Expressing, Plasmid Preparation, Luciferase, Activity Assay, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: Repressors of Androgen and Progesterone Receptor Action

doi: 10.1074/jbc.m305153200

Figure Lengend Snippet: FIG. 10. SMRT and DAX-1 bind PR and AR without removing them from the promoter. Hela cells were transfected with GRE2 E1b Luc reporter, CMV GRE3 CAT reporter, and 10 ng of AR and increasing concentrations of DAX-1 (A); 10 ng of PR and increasing concentrations of DAX-1 (B); 10 ng of AR and increasing concentrations of SMRT (C); 10 ng of PR and increasing concentrations of SMRT (D). Cells were either left untreated (open bars) or treated with the appropriate agonist for each receptor (filled bars, 3 nM R1881 for AR and 10 nM R5020 for PR). Cells were harvested, lysed, and assayed for CAT and luciferase activity, which was normalized for protein levels. Simultaneous measurements of CAT and luciferase activity show that amounts of DAX-1 that are optimal for AR and PR repression (A and B) do not take off receptors from the identical binding sites between the CMV promoter and the CAT coding sequence.

Article Snippet: Trichostatin A was obtained from Sigma Aldrich, mifepristone (RU486) from Siniwest Holdings (San Diego, CA), Methyltrienolone (R1881) and Promegestone (R5020) from PerkinElmer Life Sciences (Boston, MA).

Techniques: Transfection, Luciferase, Activity Assay, Binding Assay, Sequencing

Journal: Cell reports

Article Title: The mTOR chromatin-bound interactome in prostate cancer.

doi: 10.1016/j.celrep.2022.110534

Figure Lengend Snippet: Figure 1. RIME identification of mTOR CIPs in PCa cells (A) Schematic of mTOR RIME analysis in four PCa models treated with vehicle (DMSO), the synthetic androgen R1881, and/or mTOR inhibitor Torin 1 in biological triplicates. Vehicle-treated immunoglobulin G controls were also used. (B) mTOR protein structure and cumulative peptide coverage from mTOR RIME analysis across triplicate experiments in four PCa cell lines. (C) Total mTOR RIME CIPs identified and whether they were previously known. (D) Immunoblot analysis of mTOR, AR full-length (AR-FL) and splice variant (AR-V7), and PTEN levels in nuclear PCa homogenates. Lamin B1 levels are shown as a loading control. (E) Overlap of mTOR RIME datasets between PCa cell lines/conditions. See also Figure S1.

Article Snippet: For qRT-PCR experiments involving AR signaling inhibition, LNCaP cells were cultured as described above and hormone-deprived cells were treated with 10 nM R1881 (Cat# E3164-000, Steraloids) ± 10 mM Enzalutamide (Cat# A3003, APExBIO), or 1% DMSO as vehicle control for 48 h. For qRT-PCR experiments involving mTOR signaling inhibition, LNCaP cells were cultured as described above and hormonedeprived cells were treated with 10 nM R1881 (Cat# E3164-000, Steraloids) ± 100 nM Torin 1(Cat# T548700, Toronto Research Chemicals), or 1% DMSO as vehicle control for 48 h. For NUP210 siRNA-mediated knockdown experiments, LNCaP and PC3 cells were cultured in RPMI-1640medium containing 2% CSS for 48 h, and then trypsinized, re-seeded with CSS medium, and transfected with a pool of siRNAs (100 nM, ON-TARGETplus siRNA pool) targeting human NUP210 (Cat# L-017529-01-0005, Dharmacon/Horizon Discovery) or scrambled siRNA as a control (Cat# D-001810-10-20, Dharmacon/Horizon Discovery) with HiPerFect (Cat# 301707, Qiagen) transfection reagent per the manufacturer’s.

Techniques: Western Blot, Variant Assay, Control

Journal: Cell reports

Article Title: The mTOR chromatin-bound interactome in prostate cancer.

doi: 10.1016/j.celrep.2022.110534

Figure Lengend Snippet: Figure 2. Effect of R1881 and/or Torin 1 on chromatin mTOR-protein interactions (A) Overlap of drug-modulated mTOR CIPs from triplicate RIME experiments in AR+ LNCaP and 22Rv1 cells. (B) R1881-modulated chromatin-bound mTOR-protein affinities. Gene names for a subset of the CIPs are shown. Red, enhanced/gained interactions; blue, reduced/lost interactions. (C) AR protein structure and cumulative peptide coverage from mTOR RIME analysis across triplicate experiments in AR+ cells.

Article Snippet: For qRT-PCR experiments involving AR signaling inhibition, LNCaP cells were cultured as described above and hormone-deprived cells were treated with 10 nM R1881 (Cat# E3164-000, Steraloids) ± 10 mM Enzalutamide (Cat# A3003, APExBIO), or 1% DMSO as vehicle control for 48 h. For qRT-PCR experiments involving mTOR signaling inhibition, LNCaP cells were cultured as described above and hormonedeprived cells were treated with 10 nM R1881 (Cat# E3164-000, Steraloids) ± 100 nM Torin 1(Cat# T548700, Toronto Research Chemicals), or 1% DMSO as vehicle control for 48 h. For NUP210 siRNA-mediated knockdown experiments, LNCaP and PC3 cells were cultured in RPMI-1640medium containing 2% CSS for 48 h, and then trypsinized, re-seeded with CSS medium, and transfected with a pool of siRNAs (100 nM, ON-TARGETplus siRNA pool) targeting human NUP210 (Cat# L-017529-01-0005, Dharmacon/Horizon Discovery) or scrambled siRNA as a control (Cat# D-001810-10-20, Dharmacon/Horizon Discovery) with HiPerFect (Cat# 301707, Qiagen) transfection reagent per the manufacturer’s.

Techniques:

Journal: Cell reports

Article Title: The mTOR chromatin-bound interactome in prostate cancer.

doi: 10.1016/j.celrep.2022.110534

Figure Lengend Snippet: Figure 3. Functional enrichment analysis of mTOR CIPs in LNCaP cells ± R1881 (A) Venn diagrams showing the number of mTOR CIPs identified in LNCaP cells from biological triplicates under vehicle or R1881 conditions versus immuno- globulin G control with 87% of the total having a known nuclear localization.

Article Snippet: For qRT-PCR experiments involving AR signaling inhibition, LNCaP cells were cultured as described above and hormone-deprived cells were treated with 10 nM R1881 (Cat# E3164-000, Steraloids) ± 10 mM Enzalutamide (Cat# A3003, APExBIO), or 1% DMSO as vehicle control for 48 h. For qRT-PCR experiments involving mTOR signaling inhibition, LNCaP cells were cultured as described above and hormonedeprived cells were treated with 10 nM R1881 (Cat# E3164-000, Steraloids) ± 100 nM Torin 1(Cat# T548700, Toronto Research Chemicals), or 1% DMSO as vehicle control for 48 h. For NUP210 siRNA-mediated knockdown experiments, LNCaP and PC3 cells were cultured in RPMI-1640medium containing 2% CSS for 48 h, and then trypsinized, re-seeded with CSS medium, and transfected with a pool of siRNAs (100 nM, ON-TARGETplus siRNA pool) targeting human NUP210 (Cat# L-017529-01-0005, Dharmacon/Horizon Discovery) or scrambled siRNA as a control (Cat# D-001810-10-20, Dharmacon/Horizon Discovery) with HiPerFect (Cat# 301707, Qiagen) transfection reagent per the manufacturer’s.

Techniques: Functional Assay, Control

Journal: Cell reports

Article Title: The mTOR chromatin-bound interactome in prostate cancer.

doi: 10.1016/j.celrep.2022.110534

Figure Lengend Snippet: Figure 7. Androgens promote assembly of an mTOR-AR-HDAC2 transcriptional complex (A) Co-IP experiments in LNCaP cells show that mTOR and AR interact with NuRD complex components. (B) R1881 increases the overlap of mTOR, AR, and HDAC2 ChIP-seq peaks in PCa cells. (C) Genome-wide mapping of mTOR-AR-HDAC2 co-occupied sites between vehicle (EtOH)- and R1881-treated PCa cells. (D) Pie charts showing that most of the mTOR-AR-HDAC2 peaks found within ±20 kb of ARG TSSs are up-regulated by androgens. (E) Genome browser views showing R1881-mediated de novo formation of an mTOR-AR-HDAC2 complex at ARGs in PCa cells. (F) Heatmaps of ChIP-qPCR enrichment values in LNCaP cells showing the temporal R1881-mediated co-recruitment of mTOR, AR, NuRD-associated HDAC2 and CHD4, RNA polymerase II, and deposition of the active histone mark H3K27ac at mTOR-AR-HDAC2-targeted ARGs shown in (E). (G) Immunoblots showing efficacy of shRNA-mediated silencing of HDAC2 in LNCaP cells. Lamin B1 levels are shown as a loading control.

Article Snippet: For qRT-PCR experiments involving AR signaling inhibition, LNCaP cells were cultured as described above and hormone-deprived cells were treated with 10 nM R1881 (Cat# E3164-000, Steraloids) ± 10 mM Enzalutamide (Cat# A3003, APExBIO), or 1% DMSO as vehicle control for 48 h. For qRT-PCR experiments involving mTOR signaling inhibition, LNCaP cells were cultured as described above and hormonedeprived cells were treated with 10 nM R1881 (Cat# E3164-000, Steraloids) ± 100 nM Torin 1(Cat# T548700, Toronto Research Chemicals), or 1% DMSO as vehicle control for 48 h. For NUP210 siRNA-mediated knockdown experiments, LNCaP and PC3 cells were cultured in RPMI-1640medium containing 2% CSS for 48 h, and then trypsinized, re-seeded with CSS medium, and transfected with a pool of siRNAs (100 nM, ON-TARGETplus siRNA pool) targeting human NUP210 (Cat# L-017529-01-0005, Dharmacon/Horizon Discovery) or scrambled siRNA as a control (Cat# D-001810-10-20, Dharmacon/Horizon Discovery) with HiPerFect (Cat# 301707, Qiagen) transfection reagent per the manufacturer’s.

Techniques: Co-Immunoprecipitation Assay, ChIP-sequencing, Genome Wide, ChIP-qPCR, Western Blot, shRNA, Control

Journal: Environmental Health Perspectives

Article Title: Endocrine-Disrupting Potential of Bisphenol A, Bisphenol A Dimethacrylate, 4- n -Nonylphenol, and 4- n -Octylphenol in Vitro : New Data and a Brief Review

doi: 10.1289/ehp.9368

Figure Lengend Snippet: AR antagonism of the test chemicals on co-exposure with R1881 in transient transfected CHO-K1 cells. Cells were transiently transfected with the AR expression pSVAR0 and the reporter pMMTV-LUC vector.

Article Snippet: Methyltrienolone (R1881) was purchased from Mikromol Gmbh (Luckenwalde, Germany).

Techniques: Transfection, Expressing, Plasmid Preparation