wnt reporter construct topflash  (Upstate Biotechnology Inc)

Journal: Cell Death & Disease

Article Title: Alternative splicing of BAZ1A in colorectal cancer disrupts the DNA damage response and increases chemosensitization

doi: 10.1038/s41419-024-06954-6

Figure Lengend Snippet: A Immunoblotting in HCT116 cells after stable transfection with scrambled shCtrl or three different shRNAs targeting BAZ1A, with β-Actin as loading control. B RT-qPCR analysis of BAZ1A , CTNNB1 , CCAR2 , and c- MYC in shCtrl and shBAZ1A cells. C shCtrl and shBAZ1A KD cells were transiently transfected with TCF/LEF-responsive luciferase reporter construct TOPFlash or negative control FOPFlash vectors, along with Renilla pRL-TK plasmid to correct for transfection efficiency. D BAZ1A interactions on c- MYC were examined using ChIP assays in parental HCT116 CCAR2 +/+ or HCT116 CCAR2 -/- cells, with IgG serving as negative control. Statistical significance determined by Student’s t-test for n = 3 replicates, indicated by ***p < 0.001, ****p < 0.0001 vs. shCtrl.

Article Snippet: BAZ1A KD cells (see above) were transiently transfected with 1 μg of plasmid DNA for the Wnt reporter construct TOPflash, or the negative control FOPflash (Upstate Biotechnology, Lake Placid, NY, USA).

Techniques: Western Blot, Stable Transfection, Control, Quantitative RT-PCR, Transfection, Luciferase, Construct, Negative Control, Plasmid Preparation

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: PLoS ONE

Article Title: Expression of serine/threonine protein kinase SGK1F promotes an hepatoblast state in stem cells directed to differentiate into hepatocytes

doi: 10.1371/journal.pone.0218135

Figure Lengend Snippet: A , Phosphorylation of β-catenin in B-13 cells in response to AdV-SGK1F infection. B-13 cells were repeatedly infected with AdV-SGK1F or the control AdVs (AdV-GFP, AdV-null) at an MOI of 5 over a 4 day period (left panel) or treated with the indicated concentration of DEX for 14 days (right panel) before expression levels of the indicated proteins were determined by Western blot (20μg cell protein/lane). Data typical of 3 separate experiments. B , Infecting B-13 cells with AdV-SGK1F leads to reduction in Tcf/Lef (Wnt signalling) transcriptional activity. Topflash is a construct containing two sets (with the second set in the reverse orientation) of three copies of the TCF-binding site upstream of a thymidine kinase promoter and luciferase open reading frame. Fopflash is an identical vector to Topflash, but with mutated TCF response elements . Twenty four hours after transfection, cells were either infected with the indicated AdV at an MOI of 5 (left panel only) or treated with DEX or vehicle control as indicated (right panel). After a further 24 hours, cells were lysed and the levels of luciferase and renilla determined as outlined in the methods section. Data are the mean and SD of 5 separate determinations from the same experiment, typical of 3 separate experiments. Significantly different versus *Fopflash or # AdV-GFP infected cells using the Student’s T test (two tailed).

Article Snippet: Wnt signalling reporter constructs “Topflash” and “Fopflash” were obtained from Addgene and used as previously described [ ] Human liver tissue for hepatocyte isolation was ethically obtained via the Newcastle Biobank ( https://www.ncl.ac.uk/biobanks/ ) with over-arching ethical approval from the Newcastle & North Tyneside 1 Research Ethics Committee.

Techniques: Phospho-proteomics, Infection, Control, Concentration Assay, Expressing, Western Blot, Activity Assay, Construct, Binding Assay, Luciferase, Plasmid Preparation, Transfection, Two Tailed Test

Journal: Oncotarget

Article Title: Inhibition of FAK kinase activity preferentially targets cancer stem cells

doi: 10.18632/oncotarget.18517

Figure Lengend Snippet: (A-C) FAK inhibition reduces reporter activity of Wnt/β-Catenin but not that of Notch or Sonic Hedgehog. (A) SUM159 cells were transfected with TOP/Flash β-Catenin/TCF, Notch (CSL) or Sonic Hedgehog reporter constructs and treated with VS-4718 (10μM) in matrigel for 24 h. Luciferase activity was measured by OneGlo assay (Promega). (B) MDA-MB-231 cells were infected with TOP/Flash reporter lentivirus and briefly selected with puromycin. Reporter cells were plated in matrigel and treated with VS-4718 or VS-6063 and stimulated with Wnt3A for 24h. OneGlo luciferase assay was performed. (C) Reporter cells, as in (B) , were transfected with siRNA against FAK or control, plated in matrigel and stimulated with Wnt3A for 24h. OneGlo luciferase assay was performed. (D) VS-4718 inhibits the expression of β-catenin/TCF target genes. SUM159 cells were treated with VS-4718 at indicated concentrations in matrigel for 2 days. Total RNA was subjected to RT-PCR for the indicated genes. (E) VS-4718 inhibits p-AKT, p-GSK3β and tyrosine phosphorylation of β-catenin. SUM159 cells were grown as tumorspheres for 3 days and then treated with VS-4718 and simultaneously stimulated with either Bio or LiCl for 4h. Cell lysates were analyzed by immunoblotting for indicated proteins. (F) Non-degradable β-catenin overcomes the CSC-targeting inhibitory effect of VS-4718. MCF7 and SUM159 cells were transfected with a control plasmid or a plasmid encoding T41A mutant form of β-catenin that is non-degradable. Stable cell lines were established after G418 antibiotic selection. Cells were treated with VS-4718 in matrigel for 4 days and Aldefluor assay was carried out. (G) Inhibition of FAK kinase activity targets CSC self-renewal through β-Catenin dependent mechanism. Data presented in this figure are representative of 2 or 3 independent experiments.

Article Snippet: Luciferase reporter constructs (Wnt/β-Catenin TOPFLASH, Notch, and Hedgehog) were purchased from Qiagen and transfected using Superfect reagent (Qiagen).

Techniques: Inhibition, Activity Assay, Transfection, Construct, Luciferase, Infection, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Plasmid Preparation, Mutagenesis, Stable Transfection, Selection