Fig 1: Reduction of AR protein stability by AIL.(a) LNCaP and 22RV1 cells were treated with cycloheximide (CHX) with or without AIL for various lengths of time. AR protein level was measured by western blotting analysis. (b) LNCaP cells were cultured in c-FBS for 5 days and treated with AIL in the absence or presence of 10 nM R1881 for 12 h. Total RNA was extracted and quantitative-PCR was performed. The AR-V7 mRNA level was detected in 22RV1 cells that were treated with the indicated concentrations of AIL for 12 h. The expression of AR, AR-V7 and PSA was normalized to ß-actin expression. Data were expressed as mean±s.d. of three independent assays; Student's t-tests were performed; *P<0.05. (c) LNCaP and 22RV1 cells were treated with various concentrations of AIL with or without MG132 (10 µM) and AR protein level was measured by western blotting analysis. (d) Immunoprecipitation (IP) was done using anti-AR and immunoblotting performed with an anti-ubiquitin antibody. Input: immunoblot of lysates probed with AR antibody. (e) 22RV1 cells were treated with or without AIL in the presence of MG132. IP was done using anti-AR, anti-HSP90 and anti-HSP70 antibodies and immunoblotting (IB) was done with anti-AR, anti-HSP90, anti-HSP70 and anti-HSP40 antibodies. (f) HSP90a activity was measured by fluorescence polarization binding assay using FITC-geldanamycin in the presence of AIL or 17-AAG (positive control).
Fig 2: Interaction of AIL with p23 protein in vitro.(a) 22RV1 cells were treated with or without 1 µM AIL for 12 h. Anti-HSP90 IP was done and co-immunoprecipitated proteins were detected using indicated antibodies. (b) The interaction between p23 protein and AIL was measured by ProteOn XPR36. (c) Mapping AIL binding site on p23. (d) Top: expression level heatmap (log2 based) of AR1–651 induced genes which were inhibited by AIL. LNCaP cells were treated as described in Methods and the RNA samples of the indicated groups were sent for RNA-seq. First gene expression values (RPKM) were normalized (z-score transformed) across samples. Then the K-means clustering method was used to portion all genes into the clusters with Pearson correlation as the metric of distance. In the heat map, yellow means ‘higher' expression and blue means ‘lower' expression. Refseq IDs were converted into gene symbols listed in Supplementary Data 1. Bottom: Gene Ontology (GO) analysis of the AR1–651 target genes which inhibited by AIL. (e) Schematic illustrating the mechanism of downregulating AR protein level by AIL. When treating with AIL, the interaction of p23 and HSP90 is prevented and the interaction between AR and the molecular-chaperones is decreased, causing ubiquitination of AR. Then, AR is degraded by the proteasome, which reduces the expression of AR target genes and inhibits PCa growth and metastasis.
Fig 3: Downregulation of AR protein levels in vitro and in vivo by AIL.(a) LNCaP, 22RV1, LNCaP-MDV3100-R and VCaP cells were treated with indicated concentrations of AIL for 12, 24 and 36 h. Then cells were lysed and AR protein level was measured by western blotting analysis. (b) LNCaP, 22RV1 and c4-2b cells were treated for 12 h with indicated concentrations of AIL with or without R1881 and the AR protein level was measured by western blotting analysis. (c) AR null PC3 cells were transfected with AR-GFP in serum-free conditions for 24 h and treated with DMSO (control), R1881 (10 nM) or combined AIL (0.4 µM) and R1881 (10 nM). AR-GFP images were taken at 2 or 10 h after treatment. Five pictures were randomly selected and the GFP-AR fluorescence in cytoplasm and nucleus was quantitated using Image-Pro Plus 4.5 software (Media Cybernetics, Silver Spring, USA). The arrows indicate the location of AR-GFP. Data were expressed as mean±s.d.; Student's t-tests were performed; *P<0.05, **P<0.01. Scale bars, 10 µm. (d) LNCaP and 22RV1 cells were treated with the indicated concentrations of AIL for 12 and 24 h. Cells were lysed and AR, HSP90 and HSP70 protein levels were measured by western blotting analysis. (e) Four representative tumour samples per group were lysed. AR, HSP90, HSP70 and HSP40 protein levels were measured by western blotting analysis. (f) The mRNA levels of PSA, TMPRSS2, total AR and AR-V7 were measured by quantitative-PCR and normalized to GAPDH. The sequences of quantitative-PCR primers were listed in Supplementary Table 2. Data were expressed as mean±s.d.; two-way ANOVA followed by Bonferroni multiple comparison test were performed; ***P<0.001. (g) Photographs of xenografts treated i.p. with DMSO (control group), 10 mg kg-1 MDV and 2 mg kg-1 AIL with corresponding IHC for AR and Ki67. Scale bars, 20 µm.
Supplier Page from BPS Bioscience, Inc. for HSP90α, His-tag Recombinant