Journal: Human molecular genetics

Article Title: Expression of tumor-promoting Cyr61 is regulated by hTRA2-β1 and acidosis.

doi: 10.1093/hmg/ddr128

Figure Lengend Snippet: Figure 4. Cyr61 alternative splicing altered by splicing factor hTRA2-beta1. (A) Expression profiles of Cyr61 mRNA isoforms in HeLa, Ishikawa, SK-OV-3 and MCF-7 cells under (C) normal conditions (control), (1) after transient transfection with hTRA2-beta1 expression plasmid (48 h), and (2) hTRA2-beta1 knock- down mediated by transient transfection with hTRA2-beta1 shRNA (SCBT; 48 h). Overexpression of hTRA2-beta1 had no influence on Cyr61 alternative spli- cing. In contrast, hTRA2-beta1 down-regulation caused a marked shift of Cyr61 alternative splicing pattern toward the expression of the intron 3-free Cyr61 mRNA isoform (481 bp). Expression of 18 S RNA served as a comparative value. RT–PCR, triplicate experiments. (B) The efficacy of hTRA2-beta1 knock- down and overexpression experiments was proven by detection of significant decrease or increase in the hTRA2-beta1 protein. hTRA2-beta1 protein expression under (2) shRNA-mediated hTra2-beta1 (SCBT) knock-down, (+) hTRA2-beta1 overexpression induced by transient transfection with hTRA2-beta1 expression plasmid (courtesy of S. Stamm), and as a control (C) under regular conditions and in cancer cell lines HeLa, Ishikawa, SK-OV-3 and MCF-7. Immu- nostaining of the hTRA2-beta1 protein with rabbit IgG primary hTRA2-beta1 Ab (courtesy of S. Stamm), and COX IV protein expression as comparative value, immunostained with COX IV mAb (#3E11, Cell Signalling). Western blot. Dashed lines indicate origin from different gels.

Article Snippet: For transient transfections, cells were plated at density of 5 × 105 cells per 10 cm2 flask right before transfection. hTRA2-beta1 expression plasmid as well as TRA2-beta1 shRNA plasmid and control shRNA plasmid A [Santa Cruz Biotechnology, Inc. (SCBT)] were transfected with SatisFectionTM Transfection Reagent (STRATAGENEw, Agilent Technologies) following the manufacturer’s protocol.

Techniques: Alternative Splicing, Expressing, Control, Transfection, Plasmid Preparation, Knockdown, shRNA, Over Expression, Reverse Transcription Polymerase Chain Reaction, Western Blot

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