Journal: Cancers

Article Title: Acetyl-CoA Counteracts the Inhibitory Effect of Antiandrogens on Androgen Receptor Signaling in Prostate Cancer Cells

doi: 10.3390/cancers14235900

Figure Lengend Snippet: Effect of ACLY inhibition on the transcriptional activity of AR-FL and AR-V7. ( A ) Lysates from parental AR-negative and transformed PC-3 cells expressing the indicated constructs were subjected to Western blotting. ( B ) Parental PC-3 cells or PC-3 cells expressing either full-length AR or AR-V7 were transfected with pGL3-AR-luc and phRL-TK (Renilla) plasmids. The cells were cultured under androgen-depleted conditions (RPMI 1640 medium supplemented with 10% charcoal-stripped FBS) for 24 h followed by treatment with different combinations of BMS 303141 (BMS) (30 μM), enzalutamide (Enz) (2.5 μM), and the synthetic androgen agonist R1881 (1 nM) for 8 h. Samples were assayed for firefly and renilla luciferase activities using the Dual-Glo Luciferase assay. Values were normalized to Renilla activities. ( C ) PC-3 cells expressing either full-length AR or AR-V7 were transfected with pGL3-AR-luc and phRL-TK plasmids. The cells were cultured under androgen-depleted conditions for 24 h followed by treatment with different combinations of BMS 303141 (BMS) (30 μM), acetyl-CoA (0.1 mM), and R1881 (1 nM) for 8 h. Samples were assayed as described in B. Columns, means of three different experiments; bars, SDs. ** p < 0.001; *** p < 0.0001; ns—non-significant. Full size blots of are shown in .

Article Snippet: ORF of the AR was amplified with specific primers using EGFP-AR (plasmid #111215, Addgene, Watertown, MA, USA) as a template; ORF of the AR-V7 splice variant was amplified with specific primers using a AR-V7-pcw107 vector (plasmid #64635, Addgene, Watertown, MA, USA) as a template.

Techniques: Inhibition, Activity Assay, Transformation Assay, Expressing, Construct, Western Blot, Transfection, Cell Culture, Luciferase

Journal: Annals of Translational Medicine

Article Title: Restoration of FKBP51 protein promotes the progression of castration resistant prostate cancer

doi: 10.21037/atm.2019.11.127

Figure Lengend Snippet: Transcriptional re-activation of FKBP51 depends on AR-V7, not AR-FL in CRPC. (A) Western blot assays of AR-FL and AR-V7 expression in LNCaP-parental, LNCaP-Hormone Naïve P30 and LNCaP-AI cells. (B) Western blot detection of FKBP51 expression after AR-V7 and AR-FL overexpression in LNCaP-P30 cell, respectively. AR-FL, full length androgen receptor. (C) Western blot detection of FKBP51 expression in LNCaP-AI cell transfected with AR-V7 and AR-FL shRNA, respectively. AR-FL, full length androgen receptor. (D) AR-V7 or AR-FL overexpressed COS-1 cells were transfected with FKBP51 luciferase promoter plasmids, cultured without DHT, then, luciferase values were detected. (E) AR-V7 or AR-FL overexpressed COS-1 cells were transfected with FKBP51 luciferase promoter plasmids, cultured with DHT (10 nM), then, luciferase values were detected. (F) DNA pull down assays were used to determine the interaction of biotin-labeled FKBP51 promoter probe with AR-V7 proteins in LNCaP parental, LNCaP-P30 and LNCaP-AI cells. ***, P<0.001; NS, not significant. P, passage; CRPC, castration-resistant prostate cancer; DHT, double hydrogen testosterone.

Article Snippet: The following antibodies were used for immunofluorescence, immunoblotting and immunohistochemistry: AR-FL (Abcam,ab74272, 1:1,000 dilution for immunoblotting, 1:200 dilution for immunohistochemistry), AR-V7 (RevMAb Biosciences, 31-1109-00, 1:1,000 dilution for immunoblotting, 1:500 dilution for immunohistochemistry), FKBP51 (Abcam, Cambridge, UK, ab46002, 1:250 dilution for immunoblotting, 1:200 dilution for immunohistochemistry), NF-κB (Cell Signaling Technology, #8242, 1:1,000 dilution for immunoblotting, 1:800 dilution for immunohistochemistry), p-NF-κB (phosphor-S536) (Abcam, ab86299, 1:5,000 dilution for immunoblotting, 1:1,000 dilution for immunohistochemistry, 1:100 dilution for immunofluorescence), c-Myc (Abcam, ab56, 1:500 dilution for immunohistochemistry, 1:1,000 dilution for immunoblotting).

Techniques: Activation Assay, Western Blot, Expressing, Over Expression, Transfection, shRNA, Luciferase, Cell Culture, Labeling

Journal: Annals of Translational Medicine

Article Title: Restoration of FKBP51 protein promotes the progression of castration resistant prostate cancer

doi: 10.21037/atm.2019.11.127

Figure Lengend Snippet: AR-V7/FKBP51/NF-κB signaling axis promotes the progression of CRPC. (A) Western blot detection of indicated proteins in LNCaP-P30 cells transfected with indicated plasmids. GAPDH was used as a loading control. (B) Western blot detection of indicated proteins in LNCaP-AI cells transfected with indicated plasmids. GAPDH was used as a loading control. (C) Bioinformatic analysis of RNA-seq data from shAR-V7 and shAR-FL mediated knock-down experiments in LN95 cell lines. The RNA-seq data was retrieved fromGSE106560. Fold change of FKBP51 expression in response to AR-V7 knocked down (y axis), AR-FL knocked down (x axis) and DHT stimulation was presented. Size of green dot showed the concentration of DHT. The scales of x axis and y axis are different. P, passage; CRPC, castration-resistant prostate cancer; DHT, double hydrogen testosterone.

Article Snippet: The following antibodies were used for immunofluorescence, immunoblotting and immunohistochemistry: AR-FL (Abcam,ab74272, 1:1,000 dilution for immunoblotting, 1:200 dilution for immunohistochemistry), AR-V7 (RevMAb Biosciences, 31-1109-00, 1:1,000 dilution for immunoblotting, 1:500 dilution for immunohistochemistry), FKBP51 (Abcam, Cambridge, UK, ab46002, 1:250 dilution for immunoblotting, 1:200 dilution for immunohistochemistry), NF-κB (Cell Signaling Technology, #8242, 1:1,000 dilution for immunoblotting, 1:800 dilution for immunohistochemistry), p-NF-κB (phosphor-S536) (Abcam, ab86299, 1:5,000 dilution for immunoblotting, 1:1,000 dilution for immunohistochemistry, 1:100 dilution for immunofluorescence), c-Myc (Abcam, ab56, 1:500 dilution for immunohistochemistry, 1:1,000 dilution for immunoblotting).

Techniques: Western Blot, Transfection, Control, RNA Sequencing, Knockdown, Expressing, Concentration Assay

Journal: Annals of Translational Medicine

Article Title: Restoration of FKBP51 protein promotes the progression of castration resistant prostate cancer

doi: 10.21037/atm.2019.11.127

Figure Lengend Snippet: Level of AR-V7, FKBP51 and p-NF-κB are positively correlated in CRPC patients. (A) IHC staining of AR-V7, FKBP51, NF-κB, p-NF-κB p65 (Ser536) and c-Myc in the eleven CRPC specimens. For each sample, three different fields were randomly chosen and imaged under microscopy. Representative images were shown. (B,C) Quantitative analysis of IHC staining of the eleven CRPC tissues with Image J software. Average optical density = integrated density/area. CRPC, castration-resistant prostate cancer. The magnifications of these representative images are 10×, 20× and 40× in magnification order.

Article Snippet: The following antibodies were used for immunofluorescence, immunoblotting and immunohistochemistry: AR-FL (Abcam,ab74272, 1:1,000 dilution for immunoblotting, 1:200 dilution for immunohistochemistry), AR-V7 (RevMAb Biosciences, 31-1109-00, 1:1,000 dilution for immunoblotting, 1:500 dilution for immunohistochemistry), FKBP51 (Abcam, Cambridge, UK, ab46002, 1:250 dilution for immunoblotting, 1:200 dilution for immunohistochemistry), NF-κB (Cell Signaling Technology, #8242, 1:1,000 dilution for immunoblotting, 1:800 dilution for immunohistochemistry), p-NF-κB (phosphor-S536) (Abcam, ab86299, 1:5,000 dilution for immunoblotting, 1:1,000 dilution for immunohistochemistry, 1:100 dilution for immunofluorescence), c-Myc (Abcam, ab56, 1:500 dilution for immunohistochemistry, 1:1,000 dilution for immunoblotting).

Techniques: Immunohistochemistry, Microscopy, Software