Journal: Virus research

Article Title: The canonical Wnt/β-catenin signaling pathway stimulates herpes simplex virus 1 productive infection

doi: 10.1016/j.virusres.2018.07.020

Figure Lengend Snippet: HLF cells in 12-well plates were transfected with 0.6 μg of TOPflash or FOPflash luciferase reporter construct and 0.02 μg of the Renilla reporter construct. Renilla reporter construct was used as an internal control to allow normalization of promoter activity. At 36 h after transfection, cells were infected with HSV-1 (Panels A-D) or UV-inactivated HSV-1 (Panels E and F) at the indicated MOI. At 16 h after infection, dual luciferase activity was measured. Where indicated (Panels D and E), the designated concentration of iCRT14 was added immediately after infection. At 16 h after infection, dual luciferase activity was measured. These results are the average of three independent experiments. An asterisk denotes significant differences (P < 0.05) in promoter activity compared to the indicated control by the Student t-test.

Article Snippet: To detect the effect of HSV-1 infection on β-catenin dependent transcription, HFL, Vero, or Neuro-2 A cells were cotransfected with the Super 8x TOPflash plasmid and a plasmid encoding Renilla luciferase under the control of a minimal herpesvirus promoter (Promega) using Lipofectamine ® 3000 Transfection Reagent (Invitrogen, L3000075).

Techniques: Transfection, Luciferase, Construct, Control, Activity Assay, Infection, Concentration Assay

Journal: Virus research

Article Title: The canonical Wnt/β-catenin signaling pathway stimulates herpes simplex virus 1 productive infection

doi: 10.1016/j.virusres.2018.07.020

Figure Lengend Snippet: Vero cells were grown in 12-well dishes and then transfected with 0.4 μg of TOPflash or FOPflash luciferase reporter construct and 0.01 μg of the Renilla reporter construct. Renilla reporter construct was used as an internal control to allow normalization of promoter activity. At 36 h after transfection, cells were infected with HSV-1 (Panels A and B) or UV-inactivated HSV-1 (Panels E and F) using the indicated MOI. At 16 h after infection, dual luciferase activity was measured.

Article Snippet: To detect the effect of HSV-1 infection on β-catenin dependent transcription, HFL, Vero, or Neuro-2 A cells were cotransfected with the Super 8x TOPflash plasmid and a plasmid encoding Renilla luciferase under the control of a minimal herpesvirus promoter (Promega) using Lipofectamine ® 3000 Transfection Reagent (Invitrogen, L3000075).

Techniques: Transfection, Luciferase, Construct, Control, Activity Assay, Infection

Journal: Virus research

Article Title: The canonical Wnt/β-catenin signaling pathway stimulates herpes simplex virus 1 productive infection

doi: 10.1016/j.virusres.2018.07.020

Figure Lengend Snippet: Panel A: HLF cells were cotransfected with 0.3 μg of the TOPflash or FOPflash luciferase reporter, and 0.02 μg of Renilla reporter construct. Where indicated, 0.5 μg of β-catenin (S33Y) and 1 μg of HSV-1 VP16 construct were used to examine the effects that VP16 have on β-catenin dependent transcription in HLF cells. Neuro-2 A cells were cotransfected with 0.1 μg of the TOPflash luciferase reporter and 0.01 μg of Renilla reporter construct. Where indicated, 0.3 μg of activated β-catenin (S33Y) (Panel B) or wild type β-catenin (Panel C) and 1 μg of VP16 at the designated concentration was used to examine the effects that VP16 had on β-catenin dependent transcription in Neuro-2 A cells.

Article Snippet: To detect the effect of HSV-1 infection on β-catenin dependent transcription, HFL, Vero, or Neuro-2 A cells were cotransfected with the Super 8x TOPflash plasmid and a plasmid encoding Renilla luciferase under the control of a minimal herpesvirus promoter (Promega) using Lipofectamine ® 3000 Transfection Reagent (Invitrogen, L3000075).

Techniques: Luciferase, Construct, Concentration Assay

Journal: Frontiers in Molecular Neuroscience

Article Title: Opposite Roles of Wnt7a and Sfrp1 in Modulating Proper Development of Neural Progenitors in the Mouse Cerebral Cortex

doi: 10.3389/fnmol.2018.00247

Figure Lengend Snippet: Sfrp1 inhibits Wnt7a activity in the TOPflash luciferase reporter assay. (A) TOPflash is a luciferase reporter of β-catenin-mediated transcriptional activation with active TCF/LEF binding sites, which affect the firefly luciferase expression. The control plasmid is FOPflash , which contains mutant TCF/LEF binding sites. (B,C) After transfection of the pcDNA3.1-Sfrp1 and pcDNA3.1-Dkk1 , a statistically significant decrease in luciferase activity of Wnt1 and Wnt7a was observed in comparison with controls. Values represent mean ± SEM. n = 3, ∗∗ P < 0.01; ∗∗∗ P < 0.001; unpaired Student’s t -test.

Article Snippet: The Sfrp1 , Dkk1 coding sequences were subcloned into the pcDNA3.1 vector for the TOPflash and FOPflash luciferase reporter (Promega, United States) assay.

Techniques: Activity Assay, Luciferase, Reporter Assay, Activation Assay, Binding Assay, Expressing, Control, Plasmid Preparation, Mutagenesis, Transfection, Comparison

Journal: Cancer Science

Article Title: Discovery of chemical probes that suppress Wnt/β‐catenin signaling through high‐throughput screening

doi: 10.1111/cas.14297

Figure Lengend Snippet: A, Position frequency matrix of the T‐cell factor (TCF) motif was obtained from the JASPAR database ( http://jaspar.genereg.net ). B, TOPFlash consists of tandemly repeated TCF motifs (Wnt response elements [WREs]) upstream of a minimal promoter that drives luciferase gene expression. FOPFlash has mutated motifs (mWREs) and is used to normalize the TOPFlash activity. HAL reporter was developed as a luciferase reporter driven by 8 copies of the promoter of histidine ammonia‐lyase ( HAL ). Transcription factor (TF) that regulates the activity of HAL promoter is under investigation. C, These reporter plasmids were designed for monitoring the activity of Wnt/β‐catenin pathway in cultured cells. When the pathway is inhibited, TOPFlash and HAL reporter activities are decreased and increased, respectively

Article Snippet: Hexachlorophene , Luciferase reporter (TOPFlash) , HEK293 expressing hFz‐1 (Wnt3a‐CM) , 960 (Genesis Plus Collection, MicroSource Discovery) , β‐Catenin degradation through SIAH‐1 induction , MWT = 406.89 CLog P = 7.02708 H‐bond donors = 2 H‐bond acceptors = 2 , , .

Techniques: Luciferase, Gene Expression, Activity Assay, Cell Culture

Journal: Cancer Science

Article Title: Discovery of chemical probes that suppress Wnt/β‐catenin signaling through high‐throughput screening

doi: 10.1111/cas.14297

Figure Lengend Snippet: Inhibitors of Wnt/β‐catenin signaling discovered by high‐throughput screening (HTS)

Article Snippet: Hexachlorophene , Luciferase reporter (TOPFlash) , HEK293 expressing hFz‐1 (Wnt3a‐CM) , 960 (Genesis Plus Collection, MicroSource Discovery) , β‐Catenin degradation through SIAH‐1 induction , MWT = 406.89 CLog P = 7.02708 H‐bond donors = 2 H‐bond acceptors = 2 , , .

Techniques: HTS Assay, Luciferase, Inhibition, Fluorescence, Imaging, Drug discovery, Activation Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Over Expression, Amplified Luminescent Proximity Homogenous Assay, Expressing, Binding Assay

Journal: Cancer Science

Article Title: Discovery of chemical probes that suppress Wnt/β‐catenin signaling through high‐throughput screening

doi: 10.1111/cas.14297

Figure Lengend Snippet: A, Position frequency matrix of the T‐cell factor (TCF) motif was obtained from the JASPAR database ( http://jaspar.genereg.net ). B, TOPFlash consists of tandemly repeated TCF motifs (Wnt response elements [WREs]) upstream of a minimal promoter that drives luciferase gene expression. FOPFlash has mutated motifs (mWREs) and is used to normalize the TOPFlash activity. HAL reporter was developed as a luciferase reporter driven by 8 copies of the promoter of histidine ammonia‐lyase ( HAL ). Transcription factor (TF) that regulates the activity of HAL promoter is under investigation. C, These reporter plasmids were designed for monitoring the activity of Wnt/β‐catenin pathway in cultured cells. When the pathway is inhibited, TOPFlash and HAL reporter activities are decreased and increased, respectively

Article Snippet: KY1220 , Luciferase reporter (TOPFlash) , HEK293 (Wnt3a‐CM) , ~3599 (Chemdiv and Sigma LOPAC 1280) , Axin binding and β‐catenin degradation , MWT = 314.32 CLog P = 1.60552 H‐bond donors = 2 H‐bond acceptors = 2 , , .

Techniques: Luciferase, Gene Expression, Activity Assay, Cell Culture

Journal: Cancer Science

Article Title: Discovery of chemical probes that suppress Wnt/β‐catenin signaling through high‐throughput screening

doi: 10.1111/cas.14297

Figure Lengend Snippet: Inhibitors of Wnt/β‐catenin signaling discovered by high‐throughput screening (HTS)

Article Snippet: KY1220 , Luciferase reporter (TOPFlash) , HEK293 (Wnt3a‐CM) , ~3599 (Chemdiv and Sigma LOPAC 1280) , Axin binding and β‐catenin degradation , MWT = 314.32 CLog P = 1.60552 H‐bond donors = 2 H‐bond acceptors = 2 , , .

Techniques: HTS Assay, Luciferase, Inhibition, Fluorescence, Imaging, Drug discovery, Activation Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Over Expression, Amplified Luminescent Proximity Homogenous Assay, Expressing, Binding Assay