Journal: British Journal of Cancer

Article Title: BCAS2 promotes prostate cancer cells proliferation by enhancing AR mRNA transcription and protein stability

doi: 10.1038/bjc.2014.603

Figure Lengend Snippet: BCAS2 interacts directly with AR protein in vitro and in vivo. ( A ) BCAS2 interacts directly with AR protein in vitro . AR protein transcribed and translated in vitro was incubated with GST fusion protein. Upper panel, bound proteins were analysed by anti-AR antibodies. Lower panel, GST proteins. ( B ) The N-terminal (aa 1–139) domain of BCAS2 binds AR directly. AR protein produced by in vitro transcription/translation was incubated with the indicated GST fusion proteins and then precipitated with GST beads. The precipitated proteins were probed with the anti-AR antibody (middle panel). Lower panel, GST proteins. ( C ) Reciprocal interaction of AR and BCAS2 in vivo . 293T cells cotransfected with FLAG-BCAS2 and pSG5-AR expression plasmids were harvested for immunoprecipitation with anti-FLAG and anti-AR antibodies, respectively. ( D ) Endogenous interaction between BCAS2 and AR. Nuclear extracts of LNCaP were subjected to immunoprecipitation with anti-AR anti-BCAS2 antibodies. Bound proteins were immunoblotted with the following antibodies: mIgG: normal mouse IgG; rIgG: normal rabbit IgG; *: mouse IgG light chain, and arrow: BCAS2. ( E ) Subcellular colocalisation of BCAS2 and AR. Immunofluorescent staining of LNCaP cells, BCAS2 (green), AR (red), and nuclei (blue). Bar, 10 μ m. ( F ) Mapping the domain of AR for BCAS2 binding. Upper panel, schematic representation of the various AR deletion mutants constructed. Middle, cell extracts from various HA-tagged AR mutants were incubated with GST-BCAS2 protein. Bound proteins were subjected to western blotting analysis with anti-HA antibody. Lower panel, GST proteins. Abbreviations: CC1, coiled-coil domain 1; CC2, coiled-coil domain 2.

Article Snippet: The tissue sections were then stained with rabbit anti-BCAS2 antibody (dilution 1 : 500; Proteintech; 10414-1-AP), and the signal was detected using the SuperSensitive Polymer-HRP IHC Detection System (BioGenex, Fremont, CA, USA) according to the manufacturer's instruction.

Techniques: In Vitro, In Vivo, Incubation, Produced, Expressing, Immunoprecipitation, Staining, Binding Assay, Construct, Western Blot

Journal: British Journal of Cancer

Article Title: BCAS2 promotes prostate cancer cells proliferation by enhancing AR mRNA transcription and protein stability

doi: 10.1038/bjc.2014.603

Figure Lengend Snippet: The BCAS2 expression is significantly increased in high Gleason grade clinical samples. Paraffin-embedded tissue sections of non-neoplastic prostate and prostate cancer specimens were subjected to immunostaining with an anti-BCAS2 antibody. 3,3′-Diaminobenzidine (DAB) was used as a chromogen and hematoxylin as counterstain. Upper panel: left to right represents non-neoplastic prostate, Gleason Score=6, Gleason Score=7, and Gleason Score=8 prostate cancer sections. Lower panel: samples of 68 prostate cancer specimens and 21 non-neoplastic specimens were analysed and scored for the levels of BCAS2 expression. Statistical analysis was done by Peason's chi-square test. Brown, positive staining signals. Blue, hematoxylin counterstain.

Article Snippet: The tissue sections were then stained with rabbit anti-BCAS2 antibody (dilution 1 : 500; Proteintech; 10414-1-AP), and the signal was detected using the SuperSensitive Polymer-HRP IHC Detection System (BioGenex, Fremont, CA, USA) according to the manufacturer's instruction.

Techniques: Expressing, Immunostaining, Staining

Journal: British Journal of Cancer

Article Title: BCAS2 promotes prostate cancer cells proliferation by enhancing AR mRNA transcription and protein stability

doi: 10.1038/bjc.2014.603

Figure Lengend Snippet: BCAS2 enhances AR protein expression via a p53-dependent pathway. Overexpression of BCAS2 increases AR levels but decreases p53 protein, regardless of the presence of hormone. Following treatment with DHT (10 nM) or EtOH, LNCaP cells transfected with FLAG-BCAS2 were subjected to western blotting in the absence ( A ) or presence of 150 nM Dox ( B ). ( C ) Overexpression of BCAS2 increases AR and PSA mRNA levels. As shown in panel A , the levels of BCAS2, AR, and PSA mRNA were determined by quantitative PCR. ***: Student's t -test, P <0.001. ( D ) Reduction of BCAS2 decreases AR and increases p53 protein levels, regardless of the presence of hormone. ( E ) Depletion of BCAS2 expression reduces AR and PSA mRNA. **: Student's t -test, P <0.01. ( F ) The deprivation of BCAS2 reduces PSA promoter activity. Upper panel, all luciferase assays were carried out three times in independent experiments. Means±s.d. Lower panel, AR and BCAS2 protein expression. **: Student's t -test, P <0.01. ( G ) Depletion of BCAS2 decreases AR through a p53-dependent pathway. In the presence of DHT, LNCaP cells transfected with shBCAS2#1 along with shp53 or shCtrl were subjected to WB. The expression levels of AR and p53 protein quantified by UVP BioSpectrum-AC imaging software (UVP, Upland, CA, USA) were normalised to β -actin.

Article Snippet: The tissue sections were then stained with rabbit anti-BCAS2 antibody (dilution 1 : 500; Proteintech; 10414-1-AP), and the signal was detected using the SuperSensitive Polymer-HRP IHC Detection System (BioGenex, Fremont, CA, USA) according to the manufacturer's instruction.

Techniques: Expressing, Over Expression, Transfection, Western Blot, Real-time Polymerase Chain Reaction, Activity Assay, Luciferase, Imaging, Software

Journal: British Journal of Cancer

Article Title: BCAS2 promotes prostate cancer cells proliferation by enhancing AR mRNA transcription and protein stability

doi: 10.1038/bjc.2014.603

Figure Lengend Snippet: BCAS2 increases AR protein expression via a p53-independent proteasome pathway and enhances AR protein stability. ( A ) BCAS2 enhances AR protein expression in p53 null cells. H1299 cells were transiently transfected with AR along with increasing doses of FLAG-BCAS2 plasmid. ( B ) Overexpression of BCAS2 increases AR-dependent transcriptional activity. Upper, luciferase assays. Lower, WB. **: Student's t -test, P <0.01. ( C ) BCAS2 has no effect on AR nuclear translocation. Without ligand treatment, LNCaP cells overexpressing BCAS2 were harvested for N/C fractionation. Blots were probed for AR, BCAS2, PARP (for the nuclear fraction control), and α -tubulin (cytosolic fraction). Data from nuclear fractions were normalised to PARP; cytosolic fractions were normalised to α -tubulin. ( D ) BCAS2 protects AR from proteasome degradation. LNCaP cells transfected with shBCAS2#1 or shBCAS2#2 were treated with MG132 (20 μ M) or equal volume DMSO (vehicle for MG132) in the presence of DHT. ( E ) Overexpression of BCAS2 increases AR protein stability. Cells transfected with FLAG-BCAS2 were incubated with CHX (200 μ g ml −1 ) for the times indicated. The percentage of AR indicates the amount of AR at each time point, relative to the control (time 0 set as 100%). Upper panel, WB. Lower panel, the calculated regression curves. ( F ) Depletion of BCAS2 reduces AR protein stability. LNCaP cells transfected with shCtrl or two different shBCAS2 constructs in the presence of DHT were subjected to CHX-chase assay as described in panel E .

Article Snippet: The tissue sections were then stained with rabbit anti-BCAS2 antibody (dilution 1 : 500; Proteintech; 10414-1-AP), and the signal was detected using the SuperSensitive Polymer-HRP IHC Detection System (BioGenex, Fremont, CA, USA) according to the manufacturer's instruction.

Techniques: Expressing, Transfection, Plasmid Preparation, Over Expression, Activity Assay, Luciferase, Translocation Assay, Fractionation, Control, Incubation, Construct

Journal: British Journal of Cancer

Article Title: BCAS2 promotes prostate cancer cells proliferation by enhancing AR mRNA transcription and protein stability

doi: 10.1038/bjc.2014.603

Figure Lengend Snippet: BCAS2 can form complexes with HSP90–AR to stabilise AR protein. ( A ) BCAS2 can form a complex with HSP90–AR. 293T cells transfected with AR and HSP90-Myc plasmids were subjected to GST-BCAS2 precipitation assays. ( B ) Two-step co-immunoprecipitation identifies a BCAS2–AR–HSP0 complex. The procedure of two-step co-immunoprecipitation is outlined in the box in the upper panel. H1299 cells were co-transfected with HA-AR, Myc-HSP90, and FLAG-BCAS2, and cell lysates were first precipitated with anti-FLAG antibody. Samples were eluted with 3 × FLAG peptide and then immunoprecipitated with anti-HA tag antibody. ( C ) BCAS2 functions in AR protein stabilisation via HSP90 in the cytosol and nucleus. LNCaP cells transfected with FLAG-BCAS2 were treated with 17-AAG (0.2 μ M) in the presence or absence of DHT. Upper panel, AR and BCAS2 protein expression. Lower panel, quantitation of AR protein. ( D ) BCAS2 rescues both the nuclear and cytosol AR protein degradation induced by 17-AAG. As described in panel C , cells were harvested for N/C fractionation. Fraction samples were subjected to WB analysis for AR, FLAG (BCAS2), HSP90, PARP, and α -tubulin. The expression level of AR protein was quantified and normalised by α -tubulin for the cytosolic fraction and by PARP for the nuclear fraction. ( E ) Proposed models of BCAS2 increasing AR protein levels.

Article Snippet: The tissue sections were then stained with rabbit anti-BCAS2 antibody (dilution 1 : 500; Proteintech; 10414-1-AP), and the signal was detected using the SuperSensitive Polymer-HRP IHC Detection System (BioGenex, Fremont, CA, USA) according to the manufacturer's instruction.

Techniques: Transfection, Immunoprecipitation, Expressing, Quantitation Assay, Fractionation

Journal: British Journal of Cancer

Article Title: BCAS2 promotes prostate cancer cells proliferation by enhancing AR mRNA transcription and protein stability

doi: 10.1038/bjc.2014.603

Figure Lengend Snippet: BCAS2 affects the growth rate of LNCaP cells via an AR-dependent pathway and the sensitivity of LNCaP cells to a HSP90 inhibitor. ( A ) Overexpression of BCAS2 increases the growth rate of prostate cancer cells expressing AR. LNCaP cells infected with Lv-BCAS2 or Lv-GFP were then transfected with shAR or shCtrl. Upper panel, the growth rates of LNCaP cells were monitored using the MTT assay. Lower panel, AR and BCAS2 proteins. **: Student's t -test, P <0.01; ***: Student's t-test, P <0.001. ( B ) Depletion of BCAS2 reduces the growth rate. The growth rates of LNCaP cells transfected with shBCAS2#1, shBCAS#2, or shCtrl plasmids were also determined using the MTT assay. ( C ) BCAS2 RNAi can increase sensitivity to the HSP90 inhibitor. LNCaP cells infected with Lv-shBCAS2#1 or Lv-shCtrl were treated with various doses of 17-AAG. Cell proliferation was then measured using the MTT assay (left panel), and cell lysates were collected for western blotting analysis (right panel). **: Student's t -test, P <0.01.

Article Snippet: The tissue sections were then stained with rabbit anti-BCAS2 antibody (dilution 1 : 500; Proteintech; 10414-1-AP), and the signal was detected using the SuperSensitive Polymer-HRP IHC Detection System (BioGenex, Fremont, CA, USA) according to the manufacturer's instruction.

Techniques: Over Expression, Expressing, Infection, Transfection, MTT Assay, Western Blot