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M50 super 8x topflash

Manufactured by Addgene
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The M50 Super 8x TOPFlash is a laboratory equipment designed for specific experimental purposes. It serves a core function related to the analysis and manipulation of biological samples. Without making claims about its intended use, this product provides a capability for researchers to carry out their work.

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Lab products found in correlation

M51 super 8x fopflash, by Addgene (13 mentions) Dual luciferase reporter assay system, by Promega (9 mentions) M51 super 8x fopflash topflash mutant, by Addgene (5 mentions) Prl tk, by Promega (4 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions) Lithium chloride (licl), by Merck Group (3 mentions) Transit lt1 transfection reagent, by Mirus Bio (2 mentions) D 001810 10 20, by Horizon Discovery (2 mentions) Prl rluc myc, by Promega (2 mentions) L 020431 00 0005, by Horizon Discovery (2 mentions) Recombinant wnt3a, by R&D Systems (2 mentions) Prl tk luc, by Promega (2 mentions) Dual glo luciferase assay system, by Promega (2 mentions) Viafect transfection reagent, by Promega (2 mentions) Dual glo luciferase assay kit, by Promega (2 mentions) 8xgtiic luciferase, by Addgene (2 mentions) Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions) Prl sv40, by Promega (1 mentions) Recombinant mouse wnt3a, by R&D Systems (1 mentions) Luciferase reporter gene assay, by Roche (1 mentions)

41 protocols using m50 super 8x topflash

1

Wnt Signaling Activity Assay

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Cells were seeded in a 24 well plate in triplicates, and were transfected using Lipofectamine 2000 (Invitrogen, Life Technology) with either M50 Super 8x TOPFlash (Addgene plasmid #12456) or M51 Super 8x FOPFlash (Addgene plasmid #12457) reporter construct (kindly provided by Dr. Randal Moon [35 (link)]) and pRLSV40 (Promega). After 24 hours of transfection, cells were lysed and analyzed for expression of firefly and Renilla luciferase activity using the Dual-Luciferase Reporter Assay System (Promega) according to manufacturer’s instructions. The TOPFlash/FOPFlash ratio was calculated following normalization by the Renilla luciferase activity from pRLSV40.
Hayashi M., Baker A., Goldstein S.D., Albert C.M., Jackson K.W., McCarty G., Kahlert U.D, & Loeb D.M. (2017). Inhibition of porcupine prolongs metastasis free survival in a mouse xenograft model of Ewing sarcoma. Oncotarget, 8(45), 78265-78276.
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2

Luciferase Reporter Vectors for Survivin Promoter and TCF/LEF Activity

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pSRVN-Luc, the luciferase reporter vector for the activity of the human survivin promoter covering the region between nucleotides 1824 and 2800 of the human survivin gene (GenBank accession number U75285), has been described previously [45 (link)]. M50 Super 8x TOPFlash (Addgene plasmid # 12456; http://n2t.net/addgene:12456; RRID:Addgene_12456), the luciferase reporter vector for TCF/LEF-mediated transcriptional activity, and its negative control M51 Super 8x FOPFlash (TOPFlash mutant) (Addgene plasmid # 12457; http://n2t.net/addgene:12457; RRID:Addgene_12457) were gifts from Professor Randall Moon.
Or C.H., Huang C.W., Chang C.C., Lai Y.C., Chen Y.J, & Chang C.C. (2020). Obatoclax, a Pan-BCL-2 Inhibitor, Downregulates Survivin to Induce Apoptosis in Human Colorectal Carcinoma Cells Via Suppressing WNT/β-catenin Signaling. International Journal of Molecular Sciences, 21(5), 1773.
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3

Wnt Signaling Pathway Modulation in MC3T3 Cells

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MC3T3 cells (1×104 cells/well) were seeded in 24-well plates in α-MEM without ascorbate. After 24h, the cells were treated with 25% (v/v) NCM or LLC1 CM or DKK2-depleted LLC1 CM. After 24h, fresh α-MEM was added for 1 hour prior to transfection. 0.6 μL Turbofectin 8.0 (Origene) was added to 20 μL/well Opti-MEM (Gibco); the mixture was vortexed and incubated for 5 min at room temperature. 200 ng/well of M50 Super 8X TOPFlash or FOPFlash (Addgene) DNA [27 (link)] was added to the mixture which was incubated at room temperature for 1 hour. 250 μL/well of α-MEM was added to the transfection mixture which was added to the cells. After 4h, 500 μL of α-MEM with 25% (v/v) NCM, LLC1 CM or DKK2-depleted LLC1 CM with 40 ng/mL recombinant mouse Wnt-3a (R&D) were added to cells. After 36h, cells were lysed and luciferase expression was measured (Roche Luciferase Reporter Gene Assay).
Berent T.E., Dorschner J.M., Craig T.A., Drake M.T., Westendorf J.J, & Kumar R. (2019). Lung Tumor Cells Inhibit Bone Mineralization and Osteoblast Activity. Biochemical and biophysical research communications, 519(3), 566-571.
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4

Luciferase Assay for β-Catenin/TCF Transcription

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Luciferase assays were performed in HEK293 as described in Ref. [30 (link)]. Briefly, cells were transfected with β-catenin/TCF-responsive reporter TOP-FLASH (M50 Super 8x TOPFlash, Addgene, Cat#12456), together with CMV-β-gal [30 (link)] to normalize for transfection efficiency with CPRG (Roche). DNA content was kept uniform by using pBlueScript II SK (Addgene, Cat#212205).
Cells were plated in 24-well plates at a density of 5×104 cells/well. After 24 h luciferase reporter plasmid was transiently transfected using TransIT®-LT1 Transfection Reagent (Mirus). 48 h after transfection, cells were left untreated or treated with the compounds for 8 h and then harvested in Luc lysis buffer (25 mM Tris pH 7.8, 2.5 mM EDTA, 10% glycerol, 1% NP-40, 2 mM DTT). Luciferase activity was determined in a Tecan plate luminometer with freshly reconstituted assay reagent (0.5 mM D-Luciferin, 20 mM tricine, 1 mM (MgCO3)-4 mM Mg(OH)2, 2.7 mM MgSO4, 0.1 mM EDTA, 33 mM DTT, 0.27 mM CoA, 0.53 mM ATP).
Peruzzo R., Mattarei A., Azzolini M., Becker-Flegler K.A., Romio M., Rigoni G., Carrer A., Biasutto L., Parrasia S., Kadow S., Managò A., Urbani A., Rossa A., Semenzato G., Soriano M.E., Trentin L., Ahmad S., Edwards M., Gulbins E., Paradisi C., Zoratti M., Leanza L, & Szabò I. (2020). Insight into the mechanism of cytotoxicity of membrane-permeant psoralenic Kv1.3 channel inhibitors by chemical dissection of a novel member of the family. Redox Biology, 37, 101705.
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5

Wnt/β-Catenin Signaling Modulation

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1×105 cells/well were plated in 24 well plates. After 24 hrs. cells were transfected with pRL-RLuc-Myc (Promega, Madison WI) and M50 Super8x TOPflash (Addgene Plasmid # 12456) [45 (link)], plasmids at a 1:16 ratio. MDA-MB-436 were co-transfected with 500ng CD177-myc, E-cadherin or empty pcDNA3 plasmids, alone or together. H146 cells were co-transfected with 25 nM CD177 or Non-targeting siRNAs (L-020431-00-0005, D-001810-10-20; Dharmacon, Lafayette, CO). 24 hrs. after transfection, media was collected and 400 ng/mL recombinant Wnt3a (R & D Systems, Minneapolis MN) or vehicle was added and distributed back to the cells. For 67NR cells, following transfection 1×105 cells were plated in polyHEMA coated 24-well plates for 24 hrs. prior to treatment with WNT3a. Luciferase levels were measured using the Dual-Luciferase Reporter Assay System (Promega) 24 hrs. after treatment. Sequence for CD177 siRNA was as follows: ACACGUUGAUGCUCAUUGA, CAGUUCAGCAUGUGUGGAA, GCAACAACCUCGUUAACUC and GGACCACCAUUAUGACACA.
Kluz P.N., Kolb R., Xie Q., Borcherding N., Liu Q., Luo Y., Kim M.C., Wang L., Zhang Y., Li W., Stipp C., Gibson-Corley K.N., Zhao C., Qi H.H., Bellizzi A., Tao A.W., Sugg S., Weigel R.J., Zhou D., Shen X, & Zhang W. (2020). Cancer cell-intrinsic function of CD177 in attenuating β-Catenin signaling. Oncogene, 39(14), 2877-2889.
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6

Wnt Signaling Activation Assay in HEK293

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HEK293ΔFZD1-10 cells55 were seeded onto 48-well plates and the next day cells were transfected with M50 Super 8x TOPFlash (Addgene #12456), pRL-TK Luc (Promega E2241), FZD5 and empty vector. 4 h post transfection, medium was changed to starvation medium with or without WNT-3A (300 ng ml−1; Biotechne 5036-WN). 24 h after transfection, cells were analyzed by the Dual-Luciferase Reporter Assay System (Promega E1910) according to manufacturer’s instructions in white 96-well plates with the following modifications: cells were lysed in 50 µl Passive Lysis Buffer, Stop & Glo reagent was used at 0.5X and 25 µl of LARII and Stop & Glo Reagent were used for each well. Luminescence was measured using a Synergy2 microplate reader (BioTek).
Wright S.C., Kozielewicz P., Kowalski-Jahn M., Petersen J., Bowin C.F., Slodkowicz G., Marti-Solano M., Rodríguez D., Hot B., Okashah N., Strakova K., Valnohova J., Babu M.M., Lambert N.A., Carlsson J, & Schulte G. (2019). A conserved molecular switch in Class F receptors regulates receptor activation and pathway selection. Nature Communications, 10, 667.
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7

Plasmid Toolkit for Cell Signaling Research

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The plasmids used in this work were the following TOP-GFP-mCherry plasmid was a gift from Ramesh Shivdasani (Addgene #35491). M50 Super 8x TOPFlash (Addgene #12456) and M51 Super 8x FOPFlash (Addgene #12457) were a gift from Randall Moon. pCMV/mCherry, pSORE6/mCherry, pCMV/GFP, and pSORE6/GFP (which contains a tandem repeat of the response element to Oct4/Sox2 derived from Nanog promoter) were a gift from Marco Velasco Velázquez.
Sarabia-Sánchez M.A., Moreno-Londoño A.P., Castañeda-Patlán M.C., Alvarado-Ortiz E., Martínez-Morales J.C, & Robles-Flores M. (2023). Non-canonical Wnt/Ca2+ signaling is essential to promote self-renewal and proliferation in colon cancer stem cells. Frontiers in Oncology, 13, 1121787.
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8

Luciferase reporter assay for IGF2BP1

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Stable clones of HCT116, SW480, RKO, and CCD‐841 CoTr expressing scrambled shRNA control or IGF2BP1 shRNA were used in this assay. 20,000 cells were seeded in the 96‐well white plates with clear bottom. 24 h after seeding, they were transfected by lipofection with pGL4.74(hRLuc/TK) (5 ng) (Cat# E692A) and M50 Super 8x TOPFlash (0.05 μg) (Cat# 12456) or M51 Super 8xFOPPFlash (0.05 μg) (Cat# 12457) (Addgene). 24 h after transfection, the cells were treated with each drug for 24 h, and the luciferase activity was measured using the Dual‐Glo luciferase assay system (Cat# E2920) by Promega. Renilla luciferase was used for normalization.
Betson N., Hajahmed M., Gebretsadek T., Ndebele K., Ahmad H.A., Tchounwou P.B., Spiegelman V.S, & Noubissi F.K. (2022). Inhibition of insulin‐like growth factor 2 mRNA‐binding protein 1 sensitizes colorectal cancer cells to chemotherapeutics. FASEB BioAdvances, 4(12), 816-829.
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9

CRISPR-Cas9 Editing Efficiency Assay

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Editase expressing cells were transfected as described above in technical duplicates (SNAP-CDAR-S) or quadruplicates (Cas RESCUE-S). Cells were transfected with either M50 Super 8x TOPFlash (Addgene, #12456) or M51 Super 8x FOPFlash (Addgene, #12457) and with pcDNA3.1 expressing Renilla Luciferase for normalization. Samples were also transfected either empty, or with CTNNB1 T41-targeting (snap)2-gRNA or gRNA expressing plasmid (RESCUE-S), or with PPIB R7C-targeting (snap)2-gRNA or gRNA expressing plasmid (RESCUE-S), or with CTNNB1 T41-targeting NH2-gRNA or plasmid expressing no gRNA (RESCUE-S) in a 96-well format. Cells were lysed with 30 μl (SNAP-CDAR-S) or 20 μl (Cas RESCUE-S) per well of Passive Lysis Buffer (Promega) and shaken for 15 min. at room temperature. Luciferase signal was measured as described before (26 (link)) using the Dual-Luciferase® Reporter Assay System (Promega) following manufacturer's instructions with a Spark 10 M plate reader (Tecan). Briefly, 10 μl of each replicate (two of the four wells for RESCUE-S were pooled) were measured by addition of 35 μl of Luciferase Assay Reagent II (LAR II, Promega) and 35 μl of Stop & Glo® Reagent, and measured for 10 seconds, respectively. Editing yield was determined as described above. All luminescence measurements and editing yield determinations were conducted in biological triplicates.
Latifi N., Mack A.M., Tellioglu I., Di Giorgio S, & Stafforst T. (2023). Precise and efficient C-to-U RNA base editing with SNAP-CDAR-S. Nucleic Acids Research, 51(15), e84.
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10

Wnt/β-Catenin Signaling Assay

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Cells were assayed according to the manufacturer’s protocol. TOPFlash vectors were obtained from Addgene (M51 Super 8x FOPFlash/TOPFlash mutant, #12457; M50 Super 8x TOPFlash, 12456). Melanoma cells were plated to achieve 70% confluency in 6-well plates. Cells were co-transfected with pTK-RLuc (Green Renilla Luciferase) along with either Topflash or Fopflash vectors. After 48 hours, cells were harvested and luciferase activity was measured using the Dual-Luciferase® Reporter (DLR ) Assay System (Promega, E1910), where firefly luciferase signal was normalized to its corresponding Renilla Luciferase signal. TOPflash/FOPflash signal was determined from each treatment and graphed using GraphPad Prism.
Ndoye A., Budina-Kolomets A., Kugel CH I.I.I., Webster M., Kaur A., Behera R., Rebecca V., Li L., Brafford P., Liu Q., Gopal Y.N., Davies M.A., Mills G.B., Xu X., Wu H., Herlyn M., Nicastri M., Winkler J., Soengas M.S., Amaravadi R., Murphy M, & Weeraratna A.T. (2017). ATG5 mediates a positive feedback loop between Wnt signaling and autophagy in melanoma. Cancer research, 77(21), 5873-5885.
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