Journal: PLoS ONE

Article Title: miR-126&126* Restored Expressions Play a Tumor Suppressor Role by Directly Regulating ADAM9 and MMP7 in Melanoma

doi: 10.1371/journal.pone.0056824

Figure Lengend Snippet: A) Representative WB analysis of ADAM9 and MMP7 target genes (left); cell growth proliferation analysis (right). B) WB analysis of ADAM9 and MMP7 (left), cell growth proliferation (middle) and migration (right). Actin was utilized as internal loading control.

Article Snippet: ADAM9 and MMP7 silencing was performed by using 4 unique 29-mer shRNA constructs in retroviral GFP vector, #TG314947 for ADAM9, #TG311438 for MMP7 and #TR30013 for scrambled negative control (OriGene Technologies, Rockville, MD, USA). miRCURY locked-nucleic-acid (LNA) Power Inhibitor knockdown probes for miR-126 and -126* were obtained from Exiqon (Copenhagen, Denmark), (Product numbers #426717-00 and #426718-00).

Techniques: Migration

Journal: PLoS ONE

Article Title: miR-126&126* Restored Expressions Play a Tumor Suppressor Role by Directly Regulating ADAM9 and MMP7 in Melanoma

doi: 10.1371/journal.pone.0056824

Figure Lengend Snippet: Representative Western blot of A) ADAM9, MMP7 and OPN in normal human melanocytes (NHEM) and in a panel of melanoma cell lines, B) ADAM9 isoforms (left), MMP7 and OPN (middle) and corresponding secreted forms (right) in miR-126&126* versus empty vector-transduced Me665/1 and A375M cell lines. C) Representative Real-time PCR analysis of ADAM9 (left), MMP7 (middle) and OPN (right) mRNAs in the A375M cell line. The unresponsive short isoform of ADAM9 mRNA does not carry miR-126&126* binding sites in its 3′UTR. D) Relative expression values obtained by western blot analysis of ADAM9 (left), MMP7 (middle) and OPN (right) in A375M cells transfected with oligomers mimicking mature miR-126 or miR-126* vs non targeting (no Targ); GAPDH and actin were internal loading controls in RT-PCR and WB, respectively. Columns, of a minimum of two independent experiments; ** P <0.001; * P <0.05.

Article Snippet: ADAM9 and MMP7 silencing was performed by using 4 unique 29-mer shRNA constructs in retroviral GFP vector, #TG314947 for ADAM9, #TG311438 for MMP7 and #TR30013 for scrambled negative control (OriGene Technologies, Rockville, MD, USA). miRCURY locked-nucleic-acid (LNA) Power Inhibitor knockdown probes for miR-126 and -126* were obtained from Exiqon (Copenhagen, Denmark), (Product numbers #426717-00 and #426718-00).

Techniques: Western Blot, Plasmid Preparation, Real-time Polymerase Chain Reaction, Binding Assay, Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: miR-126&126* Restored Expressions Play a Tumor Suppressor Role by Directly Regulating ADAM9 and MMP7 in Melanoma

doi: 10.1371/journal.pone.0056824

Figure Lengend Snippet: A) Luciferase reporter assays performed by transfecting a Luc reporter gene (psiCHECK2) linked to 3′-UTR of ADAM9 or MMP7 or OPN or PI3KR2 in miR- versus empty vector-transduced A375M cell lines. B) Schematic presentation of predicted miR-126 and miR-126* target sites identified in the ADAM9 3′UTR (left) and relative miR-126&126*-dependent luciferase activities (right) in presence of wild-type (WT) or mutant (mut) binding sites in the-ADAM9 3′UTR. C) The same schematic representation (left) and luciferase experiments (right) carried out on MMP7 3′UTR. Columns, of minimum of 5 experiments per group; ** P <0.001; * P <0.05.

Article Snippet: ADAM9 and MMP7 silencing was performed by using 4 unique 29-mer shRNA constructs in retroviral GFP vector, #TG314947 for ADAM9, #TG311438 for MMP7 and #TR30013 for scrambled negative control (OriGene Technologies, Rockville, MD, USA). miRCURY locked-nucleic-acid (LNA) Power Inhibitor knockdown probes for miR-126 and -126* were obtained from Exiqon (Copenhagen, Denmark), (Product numbers #426717-00 and #426718-00).

Techniques: Luciferase, Plasmid Preparation, Mutagenesis, Binding Assay

Journal: PLoS ONE

Article Title: miR-126&126* Restored Expressions Play a Tumor Suppressor Role by Directly Regulating ADAM9 and MMP7 in Melanoma

doi: 10.1371/journal.pone.0056824

Figure Lengend Snippet: Western blot analyses showing the effectiveness of stable si-ADAM9, si-MMP7 and scrambled control (SCR) transduction. B) Invasion and migration assays in si-ADAM9- or si-MMP7-infected melanoma cell lines compared with scrambled control. C) Invasion (left) and migration (right) in presence of either ADAM9 or MMP7 recombinant proteins in miR-126&126*-transduced A375M cell lines compared with control cells. Columns, mean±SD of a minimum of two independent experiments; ** P <0.001; * P <0.05.

Article Snippet: ADAM9 and MMP7 silencing was performed by using 4 unique 29-mer shRNA constructs in retroviral GFP vector, #TG314947 for ADAM9, #TG311438 for MMP7 and #TR30013 for scrambled negative control (OriGene Technologies, Rockville, MD, USA). miRCURY locked-nucleic-acid (LNA) Power Inhibitor knockdown probes for miR-126 and -126* were obtained from Exiqon (Copenhagen, Denmark), (Product numbers #426717-00 and #426718-00).

Techniques: Western Blot, Transduction, Migration, Infection, Recombinant

Journal: PLoS ONE

Article Title: miR-126&126* Restored Expressions Play a Tumor Suppressor Role by Directly Regulating ADAM9 and MMP7 in Melanoma

doi: 10.1371/journal.pone.0056824

Figure Lengend Snippet: Representative WB of A) pro-HB-EGF (bottom) and relative densitometric analysis (top) in miR-126&126*- versus empty vector-transduced Me665/1 melanoma cell line treated or not with PMA. B ) pro-HB-EGF and HB-EGF-C levels in si-ADAM9- or si-MMP7-infected melanoma compared with si-scrambled control. C) Real time PCR analysis of ADAM9 and MMP7 in the same silenced cells.

Article Snippet: ADAM9 and MMP7 silencing was performed by using 4 unique 29-mer shRNA constructs in retroviral GFP vector, #TG314947 for ADAM9, #TG311438 for MMP7 and #TR30013 for scrambled negative control (OriGene Technologies, Rockville, MD, USA). miRCURY locked-nucleic-acid (LNA) Power Inhibitor knockdown probes for miR-126 and -126* were obtained from Exiqon (Copenhagen, Denmark), (Product numbers #426717-00 and #426718-00).

Techniques: Plasmid Preparation, Infection, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Folic Acid Remodels Chromatin on Hes1 and Neurog2 Promoters during Caudal Neural Tube Development *

doi: 10.1074/jbc.M110.126714

Figure Lengend Snippet: KDM6B directly regulates histone H3K27 methylation in WT cultured neurospheres. A, transfection of scrambled negative control shRNA-RFP (TR30015, OriGene) into cells from WT neurospheres and immunostaining for KDM6B; B, transfection with KDM6B shRNA-RFP and immunostaining for KDM6B; C, transfection of scrambled negative control shRNA-RFP and immunostaining for H3K27me3; D, transfection with KDM6B shRNA-RFP and immunostaining for H3K27me3. Experiments were performed in quadruplicate. A representative of 4 separate experiments is shown. These results demonstrate that H3K27 methylation is directly regulated by KDM6B in cultured neurospheres.

Article Snippet: A scrambled negative control-shRNA-RFP from OriGene (TR30015) did not silence KDM6B levels ( A ) or increase H3K27 methylation ( C ).

Techniques: Methylation, Cell Culture, Transfection, Negative Control, shRNA, Immunostaining

Journal: Oncotarget

Article Title: Altered glucuronidation deregulates androgen dependent response profiles and signifies castration resistance in prostate cancer

doi: 10.18632/oncotarget.28059

Figure Lengend Snippet: Two vector control cell lines (VC1 and VC2) and two UGDH-overexpressing lines (OE1 and OE2) were selected in the LNCaP AD background. Equal cell counts were seeded 48 hours in androgen depleted media, followed by removal and replacement with media containing DMSO (vehicle, 0 nM) or the indicated concentration of DHT. After an additional 48 hours, cells were harvested for analysis. AR-dependent genes PSA ( A ) and UGT2B17 ( B ) were analyzed by WB in whole cell lysates; ( C ) functional synthetic output of each cell line was assessed by Notch1 expression in cell lysates; ( D ) UDP-sugar pools were measured in cell lysates by LC-MS. In panels A–C, mean ± SEM is plotted for triplicate measurements. In panel D, mean ± SD is plotted for quadruplicate measurements. Statistical significance is indicated as: (a) p < 0.05 relative to VC1 at 0 nM DHT. (b) p < 0.05 relative to VC2 at 0 nM DHT. (c) p < 0.05 comparing OE1 to both VC1 and VC2 at the indicated [DHT]. (d) p < 0.05 comparing OE2 to both VC1 and VC2 at the indicated [DHT].

Article Snippet: To achieve UGDH knockdown, LNCaP AD and LNCaP CR cells were transfected with plasmids encoding shRNA targeted to UGDH or a scrambled non-targeting control (UGDH shRNA-pGFP-V-RS #TG334012 and 29-mer oligo-pGFP-V-RS #TR30008, respectively, from Origene Technologies, Rockville, MD, USA).

Techniques: Plasmid Preparation, Concentration Assay, Functional Assay, Expressing, Liquid Chromatography with Mass Spectroscopy

Journal: Oncotarget

Article Title: Altered glucuronidation deregulates androgen dependent response profiles and signifies castration resistance in prostate cancer

doi: 10.18632/oncotarget.28059

Figure Lengend Snippet: Two vector control cell lines (VC1 and VC2) and two UGDH-overexpressing lines (OE1 and OE2) were selected in the LNCaP CR background. Equal cell numbers were seeded 48 hours in androgen replete media followed by harvest of cells for analysis of gene expression ( A and B ) and UDP-sugars ( D ). For panel ( C ), equal cell counts were seeded 48 hours in androgen depleted media, followed by removal and replacement with media containing DMSO (vehicle, 0 nM) or the indicated concentration of DHT. After an additional 48 hours, cells and media were harvested for analysis. AR-dependent genes UGT2B17 (A) and FoxA1 (B) were analyzed by WB in whole cell lysates; (C) functional synthetic output of each cell line was assessed by Notch1 expression in cell lysates; (D) UDP-sugar pools were measured in cell lysates by mass spectrometry. Mean ± SEM is plotted for triplicate technical measurements; * p < 0.05 for OE1 and OE2 relative to VC1 and VC2.

Article Snippet: To achieve UGDH knockdown, LNCaP AD and LNCaP CR cells were transfected with plasmids encoding shRNA targeted to UGDH or a scrambled non-targeting control (UGDH shRNA-pGFP-V-RS #TG334012 and 29-mer oligo-pGFP-V-RS #TR30008, respectively, from Origene Technologies, Rockville, MD, USA).

Techniques: Plasmid Preparation, Expressing, Concentration Assay, Functional Assay, Mass Spectrometry

Journal: Oncotarget

Article Title: Altered glucuronidation deregulates androgen dependent response profiles and signifies castration resistance in prostate cancer

doi: 10.18632/oncotarget.28059

Figure Lengend Snippet: LNCaP AD and CR cells were selected for stable expression of a non-targeting vector control (VC1 and VC2) or a UGDH shRNA knockdown construct (KD1 and KD2). Equal cell counts were seeded 48 hours in androgen depleted media, followed by removal and replacement with media containing 10 nM DHT or DMSO (vehicle, 0 nM). After an additional 48 hours, cells were harvested for analysis by western blot and mass spectrometry. ( A ) AR-dependent genes PSA (panels a and d ) and UGT2B17 (panels b and e ) were analyzed by WB in whole cell lysates; functional synthetic output of each cell line was assessed by Notch1 expression in cell lysates (panels c and f ). Panels (a–c) depict expression data in the AD background and panels (d–f) illustrate data from the CR background. Mean ± SEM is plotted. Statistical significance is indicated on the plots as: (a) p < 0.05 for that clone, comparing 0 vs 10 nM DHT; (b) p < 0.05 relative to VC1 and VC2, 10 nM DHT; (c) p < 0.05 relative to VC1 and VC2, 0 nM DHT. ( B ) UDP-sugar pools were measured in cell lysates by LC-MS for both the AD (upper) and CR (lower) backgrounds as indicated. Mean ± SD is plotted. Statistical significance is indicated as: (a) p < 0.05 for both KD1 and KD2 relative to VC1 or VC2, 0 nM DHT. (b) p < 0.05 for both KD1 and KD2 relative to VC1 or VC2, 10 nM DHT. (c) p < 0.05 for that clone, comparing 0 and 10 nM DHT.

Article Snippet: To achieve UGDH knockdown, LNCaP AD and LNCaP CR cells were transfected with plasmids encoding shRNA targeted to UGDH or a scrambled non-targeting control (UGDH shRNA-pGFP-V-RS #TG334012 and 29-mer oligo-pGFP-V-RS #TR30008, respectively, from Origene Technologies, Rockville, MD, USA).

Techniques: Expressing, Plasmid Preparation, shRNA, Construct, Western Blot, Mass Spectrometry, Functional Assay, Liquid Chromatography with Mass Spectroscopy

Journal: Oncotarget

Article Title: Altered glucuronidation deregulates androgen dependent response profiles and signifies castration resistance in prostate cancer

doi: 10.18632/oncotarget.28059

Figure Lengend Snippet: ( A and B ) LNCaP AD cells overexpressing UGDH (B) were compared to vector control (A) and to LNCaP CR cells with UGDH knocked down ( C , Control; D , UGDH KD). Equal cell numbers were plated and treated for three days with the indicated concentrations of enzalutamide (in mM). Proliferating cells were quantified in a fluorescence plate reader using resazurin-resorufin conversion. Daily cell count was determined from a standard curve and represents the mean of four technical replicates ± SEM; * p < 0.05.

Article Snippet: To achieve UGDH knockdown, LNCaP AD and LNCaP CR cells were transfected with plasmids encoding shRNA targeted to UGDH or a scrambled non-targeting control (UGDH shRNA-pGFP-V-RS #TG334012 and 29-mer oligo-pGFP-V-RS #TR30008, respectively, from Origene Technologies, Rockville, MD, USA).

Techniques: Plasmid Preparation, Fluorescence, Cell Counting