Journal: Arthritis Research & Therapy

Article Title: Small molecule inhibitors of WNT/β-catenin signaling block IL-1β- and TNFα-induced cartilage degradation

doi: 10.1186/ar4273

Figure Lengend Snippet: Small molecule inhibitors of WNT/β-catenin signaling effectively block TCF/Lef-mediated activity of β-catenin . (A) Small molecules dose-dependently inhibit transcription factor/lymphoid enhancer-binding factor (TCF/LEF) reporter activity in HEK-293t cells, induced by the glycogen synthase kinase 3β (GSK3β) inhibitor 6-bromoindirubin 3'-oxime (BIO) (1.0 µM). Data represent the means of three independent experiments with 95% confidence intervals (CIs). (B) Metabolic activity, measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in KS483-4C3 cells, was not affected by small molecules at lower concentrations; however, at 1.0 µM (except for CGP049090) and 3.0 µM, metabolic activity was significantly decreased. Data represent the means of three independent experiments with 95% CI. (C) Treatment with 50 mM LiCl induced nuclear translocation of β-catenin. Small molecules by themselves had no effect on cellular localization of β-catenin, whereas PKF118-310 and PKF115-584 blocked LiCl-induced translocation of β-catenin to the nucleus. CGP049090 did not affect nuclear accumulation of β-catenin after LiCl treatment. A representative example of three independent experiments is shown. Scale bar represents 10 µm * P < 0.05 placed in the order relative to the order of the data points below..

Article Snippet: HEK-293t cells were seeded at 7,500 cells/cm 2 into 96-well plates (Nalge Nunc International, Penfield, NY, USA) and cultured for 24 h in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS) and 100 U of penicillin-streptomycin (Gibco/Life Technologies, Grand Island, NY, USA) prior to transfection with the TOPflash TCF/Lef luciferase reporter construct (Upstate Biotechnology/EMD Millipore, Lake Placid, NY, USA) and pRL-CMV control vector (Promega, Madison, WI, USA).

Techniques: Blocking Assay, Activity Assay, Binding Assay, Translocation 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: 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: Arthritis Research & Therapy

Article Title: Beneficial effect of resveratrol on phenotypic features and activity of osteoarthritic osteoblasts

doi: 10.1186/s13075-017-1365-2

Figure Lengend Snippet: Effect of resveratol ( RSV ) on Wnt/β-catenin signaling activity and mineralization in osteoblasts (Ob) in osteoarthritis (OA). OA osteoblasts were stimulated with increasing doses of RSV (10, 50, 100, 500 or 1000 nM) for up to 28 days. a Representative alizarin red staining of normal Ob. b Quantification of alizarin red staining as a function of time and chronic effect of RSV exposure in OA Ob ( n = 6). Cells were treated for 4 h with vehicle or increasing doses of RSV. c TOPflash activity in OA Ob in response to parental and Wnt3a conditioned medium either in the presence or absence of RSV for 24 h. Values are reported relative to values in parental samples ( n = 5 experiments). d Western blot analysis of β-catenin expression in OA Ob in response to Wnt3a in the presence of RSV. Representative of four experiments. e Western blot analysis of the effect of PD98059 on RSV-dependent β-catenin activation in OA Ob. Representative of three experiments. CTRL control

Article Snippet: Plasmid mixtures containing 2 μg TOPflash luciferase construct (Upstate Biotechnology, Lake Placid, NY, USA) and 0.05 μg RENILLA luciferase driven by the SV40 promoter (Promega, Madison, WI, USA) were transfected into cells using FuGENE 6 Transfection Reageant (Roche) according to the manufacturer’s protocol.

Techniques: Activity Assay, Staining, Western Blot, Expressing, Activation Assay, Control

Journal: Oncology Letters

Article Title: Long non-coding RNA CCAT2 promotes prostate cancer cell proliferation and invasion by regulating the Wnt/β-catenin signaling pathway

doi: 10.3892/ol.2020.11958

Figure Lengend Snippet: CCAT2-knockdown inhibits TCF7L2 via miR-217, thereby inhibiting the Wnt/β-catenin signaling pathway. PCa DU145 and PC3 cells were treated with si-NC, si-CCAT2, TCFL2, si-CCAT2+ TCFL2 or si-CCAT2 + LiCl. (A) The TOPflash assay was used to measure Wnt/β-catenin signaling activity. Western blotting was performed to detect the expression levels of TCF7L2, β-catenin, c-Myc and CyclinD1 in PCa (B and C) DU145 and (D and E) PC3 cells. Data are presented as the mean ± SD (n=3). *P<0.05 vs. si-NC; # P<0.05 vs. si-CCAT2. CCAT2, colon cancer associated transcript 2; TCF7L2, transcription factor 7 like 2; miR, microRNA; si, small interfering RNA; NC, negative control; LiCl, lithium chloride; PCa, prostate cancer.

Article Snippet: PCa cells (1×10 5 ) were co-transfected with 250 ng WNT signaling luciferase reporter constructs (TOPflash) (Shanghai GeneChem Co., Ltd.) and 25 ng Renilla luciferase vector (Promega Corporation) using Lipofectamine ® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol.

Techniques: Knockdown, TOPFlash assay, Activity Assay, Western Blot, Expressing, Small Interfering RNA, Negative Control

Journal: bioRxiv

Article Title: Anti-adipogenic properties of clock activator chlorhexidine and a new derivative

doi: 10.1101/2023.10.12.562086

Figure Lengend Snippet: (A, B) RT-qPCR analysis of CHX effect on expression of key components of Wnt signaling pathway in C3H10T1/2 cells (A), and 3T3-L1 preadipocytes (B), treated with indicated concentrations of chlorhexidine for 6 hours. Data are presented as Mean ± SD of n=3 replicates. *, **: p<0.05 and 0.01 CHX vs. DMSO by Student’s t test. (C, D) Representative images of immunoblot analysis of CHX effect (1µM) on β-catenin protein level in C3H10T1/2 cells before and after adipogenic induction for 8 days (C), with quantitative analysis normalized to HSP90 level (D). (E) Luciferase reporter assay of Wnt signaling pathway activity by a Wnt-responsive TOPFlash luciferase reporter treated with control or Wnt3a-containing media at 10% concentration. Data are presented as Mean ± SD of n=4 replicates. *, **: p<0.05 or 0.01 CHX vs. DMSO by Student’s t test.

Article Snippet: TOPFlash luciferase reporter luminescence was measured on microplate reader (TECAN infinite M200pro) and normalized to control FOPFlash activity, as previously described ( ).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Luciferase, Reporter Assay, Activity Assay, Concentration Assay

Journal: bioRxiv

Article Title: Anti-adipogenic properties of clock activator chlorhexidine and a new derivative

doi: 10.1101/2023.10.12.562086

Figure Lengend Snippet: (A) Docking conformation of CM002 within the CLOCK protein hydrophobic pocket. Crystal structure shown for CLOCK (red, surface mode) with Bmal1 (grey, cartoon mode) is based on PDB: 4f3l. (B) Predicted CM002 interactions with CLOCK protein residues within 3-4A 0 distance. (C-E) Baseline-adjusted tracing plots of average bioluminescence activity of U2OS cell line with stable expression of a Per2::dLuc reporter for 5 days (C), with quantitative analysis of clock period length (D) and cycling amplitude (E). CM002 at indicated concentrations were added at start of luciferase recording. Data are presented as Mean ± SD of n=4 replicates for each concentration tested. (F) RT-qPCR analysis of clock gene expression of C3H10T/2 cells treated by CM002 at indicated concentrations for 6 hours. Data are presented as Mean ± SD of n=3 replicates. *, **: p<0.05 and 0.01 CHX vs. DMSO by Student’s t test. (G, H) Analysis of CM002 effect on Wnt signaling activity using TOPFlash luciferase reporter activity assay under basal media (G) or Wnt3a- stimulated condition (H). Data are presented as Mean ± SD of n=4 replicates. *, **: p<0.05 or 0.01 CM002 vs. DMSO by Student’s t test.

Article Snippet: TOPFlash luciferase reporter luminescence was measured on microplate reader (TECAN infinite M200pro) and normalized to control FOPFlash activity, as previously described ( ).

Techniques: Activity Assay, Expressing, Luciferase, Concentration Assay, Quantitative RT-PCR

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