M51 super 8x fopflash

Manufactured by Addgene

Sourced in United Kingdom

The M51 Super 8x FOPFlash is a laboratory equipment product. It serves as a core function for its intended use, but a detailed description while maintaining an unbiased and factual approach is not available.

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Lab products found in correlation

M50 super 8x topflash, by Addgene (13 mentions) Dual luciferase reporter assay system, by Promega (4 mentions) Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions) Prl sv40, by Promega (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) Luciferase assay reagent 2, by Promega (1 mentions) Passive lysis buffer (plb), by Promega (1 mentions) Spark 10m plate reader, by Tecan (1 mentions) Lipofectamine ltx with plus reagent, by Thermo Fisher Scientific (1 mentions) Fugene hd transfection reagent, by Promega (1 mentions) X tremegene sirna transfection reagent, by Roche (1 mentions) Hygromycin b gold, by InvivoGen (1 mentions) Pcdna3.1 hygro expression vector, by Thermo Fisher Scientific (1 mentions) M50 super 8 topflash, by Addgene (1 mentions) 8xgtiic luciferase, by Addgene (1 mentions) Rpmi 1640 media, by Thermo Fisher Scientific (1 mentions) C myc promoter, by Addgene (1 mentions) Luciferase assay system, by Promega (1 mentions) X tremegene hp, by Roche (1 mentions) Luciferase assay kit, by Promega (1 mentions)

15 protocols using m51 super 8x fopflash

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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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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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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Wnt Signaling Pathway Activation Assay

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Cells grown in DMEM supplemented with antibiotics, antimycotics and 10% v/v FBS were seeded on uncoated six-well plates (Sigma Aldrich, #CLS3506) and grown to 90% confluence. Cells were then transfected in plain DMEM for 24 h with either 1 µg M50 Super 8x TOPFlash (Addgene, #12456) (40 (link)) or M51 Super 8x FOPFlash (Addgene, #12457) (40 (link)) reporter plasmids using Lipofectamine^® LTX with Plus™ Reagent (Invitrogen, #15338). Following transfection, cells were washed with PBS and subjected to stimulation. Luciferase activity was assayed with the Dual-Luciferase Reporter assay system (Promega, #E1910) according to the manufacturer’s instructions.

Koopmans T., Eilers R., Menzen M., Halayko A, & Gosens R. (2017). β-Catenin Directs Nuclear Factor-κB p65 Output via CREB-Binding Protein/p300 in Human Airway Smooth Muscle. Frontiers in Immunology, 8, 1086.

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Wnt Pathway Modulation Assay

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M50 Super 8x TOPFlash (plasmid #12456; Addgene) and M51 Super 8x FOPFlash (plasmid #12457; Addgene) were gifts from Randall Moon. Plasmids were transfected with FuGENEHD Transfection Reagent (Promega) according to the manufacturer's instructions. Antagomirs were transfected using X-tremeGENE siRNA Transfection Reagent (Roche Applied Science) according to the manufacturer's instructions. The cells were used for experiments 24–72 h after transfection.

Lee J.H., Dindorf J., Eberhardt M., Lai X., Ostalecki C., Koliha N., Gross S., Blume K., Bruns H., Wild S., Schuler G., Vera J, & Baur A.S. (2019). Innate extracellular vesicles from melanoma patients suppress β-catenin in tumor cells by miRNA-34a. Life Science Alliance, 2(2), e201800205.

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Stable LINC00313 Overexpression in HuCCT1 Cells

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Human LINC00313 (NR_026863.1) was synthesized and cloned into the pcDNA3.1/Hygro (+) expression vector (Invitrogen), by Eurofins (Eurofins Genomics, Germany). The sequence of the plasmid was verified by double-strand DNA sequencing. For the generation of stable LINC00313 over-expressing HuCCT1 clones, cells were transfected with pcDNA3.1-LINC00313 for 48 h. Then, transfected cells were grown, for 2 weeks, in fresh selection medium, consisting of 10% FBS/RPMI, in the presence of 650 µg/ml hygromycin B Gold (Invivogen, ant-hg-1). By using limiting dilution assay, individual clones from the stable LINC00313 over-expressing pool were seeded in 96-well plates and grown in selection medium. The same protocol was followed, in order to establish stable HuCCT1 clones over-expressing an empty vector (pcDNA3.1), which served as control for gain-of-function experiments. Human HA-tagged ACTL6A plasmid (VB211129-1064fwh) was constructed by Vectorbuilder. M50 Super 8x TOPFlash (Addgene plasmid #12456) and M51 Super 8x FOPFlash (TOPFlash mutant) (Addgene plasmid #12457) were a gift from Randall Moon. HuCCT1 or HEK293T cells were transfected with plasmid DNA, using Lipofectamine 2000 reagent (ThermoFisher Scientific, 11668019), according to the instructions by the manufacturer.

Papoutsoglou P., Pineau R., Leroux R., Louis C., L’Haridon A., Foretek D., Morillon A., Banales J.M., Gilot D., Aubry M, & Coulouarn C. (2024). TGFβ-induced long non-coding RNA LINC00313 activates Wnt signaling and promotes cholangiocarcinoma. EMBO Reports, 25(3), 1022-1054.

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Generating Mutant WNT2 Construct for Signaling Assays

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Active WNT2-V5 was a gift from Xi He (Addgene plasmid #43809; n2t.net/addgene:43809; RRID:Addgene_43809).^{36 (link)} Overlap PCR-based mutagenesis
was used to produce a 987 bp product with the c.338C>T: p.Ala113Val variant
(primer sequences in eTable 1, Supplemental Material, links.lww.com/NXG/A510). The mutant PCR product was digested with
NotI and BstXI and cloned by ligation into the active WNT2-V5 construct cut with
the same restriction enzymes. Firefly luciferase vectors, M50 Super 8x TOPFlash
(Addgene plasmid # 12456; n2t.net/addgene:12456;
RRID:Addgene_12456) and M51 Super 8x FOPFlash (Addgene plasmid # 12457;
n2t.net/addgene:12457; RRID:Addgene_12457), were gifts from
Randall Moon.^{37 (link)} Renilla
luciferase vector, pRL-TK plasmid, was obtained from Promega (Cat
#E2241).

Bennett M.F., Hildebrand M.S., Kayumi S., Corbett M.A., Gupta S., Ye Z., Krivanek M., Burgess R., Henry O.J., Damiano J.A., Boys A., Gécz J., Bahlo M., Scheffer I.E, & Berkovic S.F. (2022). Evidence for a Dual-Pathway, 2-Hit Genetic Model for Focal Cortical Dysplasia and Epilepsy. Neurology: Genetics, 8(1), e652.

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Cloning WNT8B p.L70P Mutant Plasmid

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Active WNT8B-V5 (WNT8B wild-type) was a gift from Xi He (Addgene plasmid # 43819; http://n2t.net/addgene:43819, (accessed on 15 May 2023; RRID:Addgene_43819) [43 (link)]. M50 Super 8× TOPFlash (Addgene plasmid # 12456; http://n2t.net/addgene:12456, (accessed on 15 May 2023; RRID:Addgene_12456) and M51 Super 8x FOPFlash (Addgene plasmid # 12457; http://n2t.net/addgene:12457, (accessed on 15 May 2023; RRID:Addgene_12457) were gifts from Randall Moon [44 (link)]. Renilla luciferase vector, pRL-TK plasmid, was obtained from Promega (Cat #E2241, Promega, Alexandria, Australia). The WNT8B p.L70P vector was cloned using a PCR insert, with a mutant sequence flanked by SacI and ApaI sites ligated into the Active Wnt8B-V5 vector backbone. The PCR insert was generated by combining overlapping amplicons made with the following primer pairs: pair 1, WNT8B3F_43810 5′-AAGCAGAGCTCTCTGGCTAAC-3′ (SacI underlined) and WNT8B2R_43742 5′-TGGCTGGACAGCTGCGGGGCTCTCTCAGGGC-3′ (mutant base bold, underlined); pair 2, WNT8B2F_43741 5′-GCCCTGAGAGAGCCCCGCAGCTGTCCAGCCA-3′ (mutant base bold, underlined) and WNT8B4R_43811 5′-CAGGGCCCGCCCGCGCCTTA-3′ (ApaI site underlined).

Ha T.T., Burgess R., Newman M., Moey C., Mandelstam S.A., Gardner A.E., Ivancevic A.M., Pham D., Kumar R., Smith N., Patel C., Malone S., Ryan M.M., Calvert S., van Eyk C.L., Lardelli M., Berkovic S.F., Leventer R.J., Richards L.J., Scheffer I.E., Gecz J, & Corbett M.A. (2023). Aicardi Syndrome Is a Genetically Heterogeneous Disorder. Genes, 14(8), 1565.

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Cell Line Authentication and Plasmid Acquisition

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All cell lines were from ATCC and cell line authentication was performed by ATCC. HCC827 and H2170 cells were cultured in a humidified incubator under 5% CO2 in RPMI-1640 media (Invitrogen) supplemented with 10% fetal bovine serum. Specimens were kept at 4 °C until they were ready for processing and sliced into 4 µm thick sections for immunohistochemical staining. 8xGTIIC-luciferase was obtained from Stefano Piccolo (Addgene plasmid # 34615) 27 (link), M50 Super 8x TOPFlash and M51 Super 8x FOPFlash (TOPFlash mutant) were provided by Randall Moon (Addgene plasmid # 12456) 28 (link).

Liu T., Guo W., Luo K., Li L., Dong J., Liu M., Shi X., Wang Z., Zhang J., Yin J., Qiu N., Lu M., Chen D., Jia X., Liu H., Gu Y., Xiong Y., Zheng G., Xu G., He Z, & Zhang Z. (2022). Smoke-induced SAV1 Gene Promoter Hypermethylation Disrupts YAP Negative Feedback and Promotes Malignant Progression of Non-small Cell Lung Cancer. International Journal of Biological Sciences, 18(11), 4497-4512.

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Generating CTNNB1 Missense Variants

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A pcDNA 3.1 mammalian expression vector carrying wild-type CTNNB1 coding sequence with a C-terminal V5 tag was provided by Dr. Yoshitaka Sekido.^{21 (link)} From this vector, we substituted the V5 tag for a Myc tag by PCR-based cloning. Using overlap PCR, we generated four CTNNB1 missense variants identified in individuals with NDDs, c.1163T>C:p.Leu388Pro (rs1559474140), c.1723G>A:p.Gly575Arg (rs797044875), c.1271T>G:p.Leu424Arg (rs863224864), c.2128C>T:p.Arg710Cys (rs748653573), and two predicted benign variants from gnomAD database, c.860A>G:p.Asn287Ser (rs35288908; Allele frequency [AF] 6.02E-04 ) and c.1188A>C:p.Glu396Asp (rs751375496; AF 1.77E-05). Cloning strategies of these variants are summarized in Supplementary Table 5. Successful cloning of these variants was confirmed by Sanger sequencing. M50 Super 8x TOPFlash (Addgene plasmid # 12456; http://n2t.net/addgene:12456; RRID:Addgene_12456) and M51 Super 8x FOPFlash (Addgene plasmid # 12457; http://n2t.net/addgene:12457; RRID:Addgene_12457) were a gift from Randall Moon.^{22 (link)} Renilla luciferase vector, pRL-TK plasmid was obtained from Promega (Cat #E2241).

Kayumi S., Pérez-Jurado L.A., Palomares M., Rangu S., Sheppard S.E., Chung W.K., Kruer M.C., Kharbanda M., Amor D.J., McGillivray G., Cohen J.S., García-Miñaúr S., van Eyk C.L., Harper K., Jolly L.A., Webber D.L., Barnett C.P., Santos-Simarro F., Pacio-Míguez M., del Pozo A., Bakhtiari S., Deardorff M., Dubbs H.A., Izumi K., Grand K., Gray C., Mark P.R., Bhoj E.J., Li D., Ortiz-Gonzalez X.R., Keena B., Zackai E.H., Goldberg E.M., de Nanclares G.P., Pereda A., Llano-Rivas I., Arroyo I., Fernández-Cuesta M.Á., Thauvin-Robinet C., Faivre L., Garde A., Mazel B., Bruel A.L., Tress M.L., Brilstra E., Fine A.S., Crompton K.E., Stegmann A.P., Sinnema M., Stevens S.C., Nicolai J., Lesca G., Lion-François L., Haye D., Chatron N., Piton A., Nizon M., Cogne B., Srivastava S., Bassetti J., Muss C., Gripp K.W., Procopio R.A., Millan F., Morrow M.M., Assaf M., Moreno-De-Luca A., Joss S., Hamilton M.J., Bertoli M., Foulds N., McKee S., MacLennan A.H., Gecz J, & Corbett M.A. (2022). Genomic and phenotypic characterization of 404 individuals with neurodevelopmental disorders caused by CTNNB1 variants. Genetics in medicine : official journal of the American College of Medical Genetics, 24(11), 2351-2366.

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