Fig 1: ATRA enhances the inhibitory response of EPI compounds.a, b AR-V7 transcriptional activity in LNCaP cells ectopically expressing AR-V7 and transfected with a reporter regulated by AR-V7-binding sites (V7BS3-luciferase). Cells were incubated with a enzalutamide (10 µM), EPI-002 (25 µM), juglone (20 µM), ATRA (10 µM), or vehicle (DMSO) for 24 h, or either b Pin1 siRNA or a non-targeting control siRNA for 48 h (n = 3 independent experiments). c, d Western blots showing the levels of ectopic AR-V7 and the efficiency of the Pin1 knockdown. The inhibitory dose–response of e EPI-002 and f EPI-7170 for inhibiting the androgen-induced PSA(6.1 kb)-luciferase activity in LNCaP cells, in the presence and absence of 5 µM of ATRA. Data shown are normalized to the fold-induction by R1881, which was approximately 82-fold at 24 h. ATRA shifted the inhibitory dose–response curves of EPI-002 and EPI-7170 to the left, thereby lowing their IC50 values. Graphs show the fractional responses of g EPI-002 and h EPI-7170 as monotherapies and combination therapy with 5 µM of ATRA, compared to the predicted additive effect determined by the Bliss independence model (n = 4). Data shown represent the means ± s.e.m. *P < 0.05, **P < 0.01; ns not significant.
Fig 2: Pin1 inhibitors block the transcriptional activity of AR.a, b Inhibitory dose–response curves for juglone, ATRA, and 13cisRA on the activity of PSA(6.1 kb)- and PB-luciferase in LNCaP cells stimulated with R1881 (1 nM) for 24 h. Data shown are normalized to the induction by R1881, which was 149-fold for PSA (6.1 kb)- and 1420-fold for PB-luciferase, n = 4 independent experiments. (c, d) Activities of CMV and AP-1 reporters after incubating with juglone (20 µM), 13cisRA (10 µM), ATRA (10 µM), or vehicle (DMSO) for 24 h. Data shown represent the means ± s.e.m. from four independent experiments. e Western blot analysis of PSA, AR, and Pin1 protein levels from LNCaP cells treated with juglone (20 µM) or ATRA (10 µM), and 1 nM of R1881 or vehicle for 24 h. f Graph showing the quantified PSA levels after normalizing to β-actin. g Representative fluorescence micrographs showing the localization of YFP-AR in LNCaP cells pre-treated with the indicated compounds and stimulated with R1881 (1 nM) or vehicle (EtOH) for 2 h. The scale bar represents 20 μm. h YFP-AR localization was quantified by calculating the ratio of average YFP intensity in the nucleus compared to the cytosol. Scores greater than 1 indicate nuclear localization. At least 50 cells were scored for each treatment. Data shown are the normalized means ± s.e.m. from three independent experiments. Statistical significance was determined by one-way ANOVA using Holm-Sidak’s multiple comparisons test. ***P < 0.001; ns not significant.
Fig 3: Pin1 is essential for the ligand-dependent transcriptional activity of AR.a Schematic depicting the position of putative Pin1 motifs on AR, which are based on experimentally verified phosphorylation sites from the Phospho.ELM database. Below, the RONN plot shows regions of predicted protein disorder where scores are above the 0.5 threshold. The numbering of residues is based on the 919 amino acid reference sequence for human AR, NCBI Accession No. AAA51729.1. DBD DNA-binding domain, H hinge region, LBD ligand-binding domain. b Western blot analysis of Pin1, DAPK1, and PLK1 expression in human prostate cancer cell lines and HEK293 cells (positive control) maintained in media supplemented with serum. c Pin1 protein levels in LNCaP cells after incubating with siRNA for 48 h. A non-targeting siRNA was used as a control. d Graph summarizing the knockdown efficiencies of the Pin1 siRNAs. e, f The activities of AR-driven reporters PSA(6.1 kb) and Probasin (PB) luciferase in LNCaP cells incubated with siRNA and androgen (R1881, 1 nM) for 48 h. g, h Activities of CMV- and AP-1-luciferase reporters which are not regulated by AR. Results shown are the means ± s.e.m. from three independent experiments. Statistical significance was determined by two-way ANOVA using Dunnett’s multiple comparisons test. **P < 0.01, ***P < 0.001; ns, not significant.
Fig 4: Pin1 interacts with the AR NTD and regulates transactivation.a, b Transactivation assays performed in LNCaP cells expressing a vector encoding the human AR NTD fused to a Gal4 DNA-binding domain (Gal4-ARN). Cells were treated with vehicle (DMSO), EPI-002 (25 μM), juglone (20 μM), or ATRA (10 μM), and then incubated with IL-6 (a 50 ng/mL) or forskolin (b 25 μM) for 24 h, n = 8 independent experiments. c, d Gal4-ARN activity in cells incubated with Pin1 siRNA or a non-targeting control siRNA and then stimulated with IL-6 or forskolin for 24 h, n = 3. Transactivation assays performed using (e) Gal4-ARN fragments (n = 6), or (f) Gal4-AR234–391 constructs (n = 3) carrying proline to glycine mutations, and treated with juglone (20 µM) and IL-6 (50 ng/mL) for 24 h. Pull-down assays performed on LNCaP cells transfected with an expression vector encoding a polyhistidine-tagged (g) AR NTD or (h) AR234–391 construct and then incubated with IL-6 (50 ng/mL) for 6 h. Input samples show Pin1 expression from lysates before the pull-down. i Co-immunoprecipitation assay showing AR protein co-immunoprecipitated with STAT3 from LNCaP cells incubated with EPI-002 (EPI, 35 μM), juglone (JUG, 30 μM), or vehicle (VEH, DMSO), and IL-6 (50 ng/mL) for 6 h. j, k Levels of phosphorylated MAPK (isoforms p44 and p42) and phosphorylated STAT3 (Tyr705 and Ser727) from LNCaP cells pre-treated with juglone (20 µM) and then stimulated with IL-6 (50 ng/mL) for 15 min. Error bars show the mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001; ns not significant.
Fig 5: Combination therapy with ATRA and EPI reduces the in vivo growth of CRPC xenografts driven by AR-Vs.a Cell cycle analysis by flow cytometry of LN95-D3 subline incubated with monotherapy and combination therapy of ATRA (5 or 10 μM) with EPI-002 (25 μM), EPI-7170 (5 μM), or enzalutamide (ENZ, 10 μM), for 48 h in media supplemented with 1.5% CSS. b Growth curves of LN95-D3 tumors established in castrated male NSG mice bearing 21-day release placebo or ATRA (5 mg) pellets and treated daily with either vehicle (CMC) or EPI-7170 (30 mg/kg/d), where pellets were implanted on day 0 and oral dosing began on day 3. c Graph showing the average body weight of animals for each treatment group during the study. d, e Images of the xenograft tumors harvested at the end of the study. Scale bars represent 5 mm (f) western blot analysis showing the expression of Pin1, Cyclin D1, Skp2, and AR from representative tumors. Quantification of the data is shown for g Pin1 and h Skp2, where the error bars represent the mean ± s.e.m. Statistical significance was determined by one- and two-way ANOVA using Dunnett’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001; ns not significant.
Supplier Page from Abcam for Recombinant human Pin1 protein (Tag Free)