The effect of Coronin 3 on Wnt/β-catenin signaling was detected using a pair of luciferase reporter constructs, as previously described.18 (link) Briefly, TOPflash and FOPflash (Millipore) were used; TOPflash contains 3 TCF/LEF sites—upstream of a thymidine kinase promoter—and a firefly luciferase gene, while the FOPflash construct contains 3 mutated TCF/LEF sites, and is used as a control for measuring nonspecific reporter activation (Promega, Madison, WI). Luciferase activity was measured using a dual-luciferase assay system kit (Promega), according to the manufacturer’s protocol, with the Renilla reniformis (sea pansy) luciferase activity as an internal control. Relative luciferase activity was expressed as normalized-fold change to controls.
Topflash
Manufactured by Merck Group
Sourced in United States, Germany
TOPflash is a laboratory equipment product. It is a tool used for various research and analysis tasks in a laboratory setting. The core function of TOPflash is to provide a reliable and accurate measurement solution for researchers and scientists.
Automatically generated - may contain errors
Lab products found in correlation
Fopflash, by Merck Group (30 mentions)
Dual luciferase reporter assay system, by Promega (13 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (13 mentions)
Prl tk, by Promega (9 mentions)
Luciferase assay system, by Promega (6 mentions)
Dual glo luciferase assay system, by Promega (6 mentions)
Fop flash plasmid, by Merck Group (3 mentions)
Lipofectamine 2000, by Merck Group (3 mentions)
Prl tk vector, by Promega (3 mentions)
Dual luciferase reporter assay, by Promega (3 mentions)
Microplate luminometer, by Berthold Technologies (3 mentions)
Varioskan lux, by Thermo Fisher Scientific (2 mentions)
Dual luciferase reporter kit, by Promega (2 mentions)
Fugene, by Roche (2 mentions)
Dual luciferase reporter assay kit, by Promega (2 mentions)
Translt1 reagent, by Mirus Bio (2 mentions)
Topflash, by Promega (2 mentions)
One glo luciferase reagent, by Promega (2 mentions)
Pfop flash, by Merck Group (2 mentions)
Lipofectamine 2000, by Promega (2 mentions)
54 protocols using topflash
1
Coronin 3 Modulates Wnt/β-catenin Signaling
2
Evaluating FOXQ1-Mediated Wnt/β-Catenin Signaling
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To assess the influence of FOXQ1 levels on Wnt/β-catenin signaling activities, a TOPFlash/FOPFlash reporter assay was performed. Cells from the Foxq1-sh mBMSC, Foxq1-over mBMSC, lv3 mBMSC, and lv5 mBMSC groups and Foxq1-over+siAnxa2 mBMSCs were seeded on 96-well plates at a density of 4 × 103 cells per well. Then, they were transiently transfected with TOPFlash or FOPFlash luciferase reporter plasmid (17-285; Millipore Sigma; MA, USA) according to the manufacturer’s protocol. The firefly luciferase activity level was normalized against the Renilla luciferase activity level. The fold increase indicating the TOPFlash activity compared to the FOPFlash is reported.
3
Quantifying Wnt/CTNNB1 Signaling
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The TOP Flash and FOP Flash luciferase reporter plasmids were purchased from MilliporeSigma company (Burlington, MA, USA). Luciferase plasmids were transiently co-transfected into U251 and U87 cells. 48 hr later, luciferase reporter assays were performed to quantify the activity of Wnt/CTNNB1 signaling pathway with dual-luciferase assay kit (Vigorous Biotech, Beijing, China) in according with protocol.
4
Wnt signaling pathway activation analysis
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HEK293T cell line was transiently cotransfected with the same amount of TOP-Flash (MilliporeSigma) and SV-40 Renilla (Promega) vectors (150 ng each vector/well of a 6-well plate) in conjunction with 200 ng/well HA-hLGR4 WT or mutated vectors. The total amount of DNA transfected was kept constant to 500 ng/well by adding the appropriate amount of pBlueScript vector. After 24 hours, cells were treated with 500 μL of conditioned medium: control, Wnt3a, Rspo1, or Wnt3a+Rspo1. After a 24-hour treatment, cells were harvested and assayed for luciferase using the Dual Luciferase Reporter System (Promega) following the manufacturer’s instructions. Each experiment was performed in triplicate and repeated 4 independent times. Samples were processed using the POLARstar Omega microplate reader, and data were analyzed using MARS Omega software.
5
Quantifying β-catenin Transcriptional Activity
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The β-catenin reporter plasmid (TOP-flash) and its mutant control (FOP-flash) were constructed by Millipore Corporation (Massachusetts, USA). Cells were serum-starved overnight and co-transfected with 0.2 μg TOP flash or FOP flash expression plasmids and 0.1 μg pRL-TK (Renilla TK-luciferase vector; Promega, Madison, USA) using Lipofectamine 2000. The activities of both firefly and Renilla luciferase reporters were determined at 48 hours after transfection using a Dual Luciferase Assay Kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The TOP-flash reporter activity is presented as the relative ratio of firefly luciferase activity to Renilla luciferase activity, and the TOP/FOP ratio was used as a measure of β-catenin-driven transcription.
6
Evaluating CTNNB1 promoter activity
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The previously reported promoter region for CTNNB1 (−2760/+27) was amplified and cloned into the pGL3 vector (Promega, Madison, WI, USA) [24 (link)]. MDA-MB-231 and MCF-7 cells were transfected with the pGL3-CTNNB1/promoter together with pIRES2-EGFP-ALX4 or control vector and pRTK-Luc (Renilla-TK-luciferase vector, Promega) to normalize the transfection efficiency. 36 h later, the activities of Firefly luciferase and Renilla luciferase were measured using the Dual Luciferase Reporter Assay System (Promega). For TOP and FOP flash assay, MDA-MB-231 and MCF-7 cells were plated into 24-well plates at a concentration of 2.0 × 104 cells per well. Cells were co-transfected with 300 ng of either TOP flash (T-cell factor reporter plasmid) or FOP flash (mutant T-cell factor reporter plasmid) expression plasmids (Millipore, Temecula, CA, USA), and 300 ng of pIRES2-EGFP-ALX4 or control vector and 30 ng pRL-TK. Luciferase activity was measured in triplicate using the fluorescence microplate reader measurement system Varioskan LUX (Thermo Fisher, Waltham, MA, USA) using a Dual-luciferase reporter kit (Promega) as previously described [25 (link)].
7
Wnt1 Signaling Pathway Regulation
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HEK293T cells were seeded in 24-well plates and transiently transfected with various amounts (0, 200 and 400 ng) of HA/SOX7, 100 ng Wnt1, TOPFlash or FOPFlash luciferase reporter plasmid (Millipore, Germany). The luciferase activity was measured using the Dual-luciferase Reporter Assay System (Promega, USA).
8
Regulation of CTNNB1 Promoter Activity
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The previously reported promoter region of full-length human β-catenin (CTNNB1) promoter (Catenin-promoter-full: -2760/+27) 32 (link) and two overlapping regions, Catenin-promoter-1 (-2760/-1161) and Catenin-promoter-2 (-1952/+27), were amplified and cloned into a pGL3-basic vector (Promega, Madison, WI, USA). LTEP-a-2 and SPC-a-1 cells were transfected with the pGL3-CTNNB1-promoters together with pIRES2-EGFP-LHX6 or control vector and pRLTK-Luc (Renilla-TK-luciferase vector, Promega) to normalize the transfection efficiency. After 36 h, the activities of firefly luciferase and renilla luciferase were measured in triplicate using the fluorescence microplate reader measurement system Varioskan LUX (Thermo Fisher, Waltham, MA, USA) with a Dual-luciferase reporter kit (Promega) as previously described 9 (link). For the TOP and FOP flash assay, LTEP-a-2 and SPC-a-1 cells were plated into 24-well plates at a concentration of 2.0×104 cells per well. Cells were co-transfected with 300 ng of either TOP flash (T-cell factor reporter plasmid) or FOP flash (mutant T-cell factor reporter plasmid) expression plasmids (Millipore, Temecula, CA, USA) and 300 ng of pIRES2-EGFP-LHX6 or control vector and 20 ng pRL-TK. Luciferase activity was detected as described above. The experiment was performed in triplicate wells for three times.
9
Wnt Pathway Reporter Assay
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For the Wnt pathway reporter assay, SW480 and HT-29 cells were seeded in 12 well plates at 3 × 105 cells/well. After 24 h, cells were transfected in serum and antibiotic-free media with 1 µg of TOP-Flash (plasmid that contains wild type TCF binding sites) or 1 µg of Fop-Flash (plasmid that contains mutated TCF binding sites), both from Millipore, using Lipofectamine® 2000 from Thermo Fisher, Waltham, MA, USA. Cells were treated 24 h after transfection with different concentrations of coffee extracts, CGA, or molecules to control the induction and inhibition of the Wnt pathway, such as CHIR 99021 and iCRT14, respectively. The luciferase activity was determined at 24 h of treatments using the Luciferase Assay System from Promega. The efficiency of transfection was determined by flow cytometry using GFP reporter plasmid. The promoter activity was expressed as the net of TOP-Flash relative light units after the substation of the associated Fop-Flash relative light units. All assays were performed in triplicate.
10
Quantification of Wnt and NFκB Signaling
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Example 21
Luciferase activity was measured using the Dual Luciferase Reporter Assay System (Promega) as previously described (Sukhdeo, K. et al. Proc Natl Acad Sci USA 104, 7516-21 (2007)). To measure Wnt or NFκB reporter activity, Colo320 cells were transfected with TOP-FLASH, FOP-FLASH plasmid (Millipore Corporation) or NFκB luciferase reporter (Stratagene), along with an internal Renilla control plasmid (hRL-null). Transfection was accomplished using FuGENE (Roche) according to the manufacturer's protocol. The results were normalized to control Renilla activity. The reported data represent the average of three independent transfection experiments performed in triplicate. See results in
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