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supertopflash  (Addgene inc)


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    Addgene inc supertopflash
    A) Mesenchymal cells harvested from the frontonasal mass of stage 24 (E4.5) embryos and were plated into high-density cultures. B) Other cultures were sectioned and used for microscopic analysis. C,E-H) There is a significant decrease in the proportion of cartilage from the DVL1 1519ΔT variant compared to the wtDVL1 infected cultures as measured in wholemount stained cultures. When the DVL1 1519* truncation was compared to wtDVL1 there was no significant difference in the Alcian blue stained area. The DVL1 1519* construct reduced cartilage compared to GFP controls. D,I-L) The cultures were significantly thinner in the presence of the DVL1 1519ΔT variant compared to w tDVL1 or the GFP controls. The DVL1 1519 * construct slightly reduced the thickness of the cartilage compared to GFP controls. M) The viruses containing human DVL1 constructs were expressed at similar levels in primary mesenchyme as determined by qRT-PCR with human-specific DVL1 primers. Generally the human DVL1 gene expression was elevated 10 to 15-fold by the viral transgenesis. N, O) The 1519ΔT virus significantly reduced expression of TWIST2 , MMP13 and LEF1 . P,Q) Two reporters were used in micromass cultures from frontonasal mass cells, the <t>SuperTOPFlash</t> reporter for canonical WNT signaling and ATF2 for JNK-PCP, non-canonical WNT signaling. The wt DVL1 plasmid significantly activated both reporters. In comparison both 1519ΔT and 1519* viruses did not activate the reporters as much as wt DVL1 . They did retain more activity than the control, parent plasmid. Statistical analysis done with one-way ANOVA followed by Dunnett’s multiple comparison test ( M ) or Tukey’s post-hoc test (C,D,N,O,P,Q). Scale bar in E-H = 2 mm, I-L = 20µm.
    Supertopflash, supplied by Addgene inc, used in various techniques. Bioz Stars score: 96/100, based on 579 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/supertopflash/M50+Super+8x+TOPFlash+(Plasmid+%2312456)/bio_rxiv__64898__2026__02__14__705933-358-3-6
    Average 96 stars, based on 579 article reviews
    supertopflash - by Bioz Stars, 2026-11
    96/100 stars

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    1) Product Images from "The abnormal C-terminus in DVL1 impacts Robinow Syndrome phenotypes"

    Article Title: The abnormal C-terminus in DVL1 impacts Robinow Syndrome phenotypes

    Journal: bioRxiv

    doi: 10.64898/2026.02.14.705933

    A) Mesenchymal cells harvested from the frontonasal mass of stage 24 (E4.5) embryos and were plated into high-density cultures. B) Other cultures were sectioned and used for microscopic analysis. C,E-H) There is a significant decrease in the proportion of cartilage from the DVL1 1519ΔT variant compared to the wtDVL1 infected cultures as measured in wholemount stained cultures. When the DVL1 1519* truncation was compared to wtDVL1 there was no significant difference in the Alcian blue stained area. The DVL1 1519* construct reduced cartilage compared to GFP controls. D,I-L) The cultures were significantly thinner in the presence of the DVL1 1519ΔT variant compared to w tDVL1 or the GFP controls. The DVL1 1519 * construct slightly reduced the thickness of the cartilage compared to GFP controls. M) The viruses containing human DVL1 constructs were expressed at similar levels in primary mesenchyme as determined by qRT-PCR with human-specific DVL1 primers. Generally the human DVL1 gene expression was elevated 10 to 15-fold by the viral transgenesis. N, O) The 1519ΔT virus significantly reduced expression of TWIST2 , MMP13 and LEF1 . P,Q) Two reporters were used in micromass cultures from frontonasal mass cells, the SuperTOPFlash reporter for canonical WNT signaling and ATF2 for JNK-PCP, non-canonical WNT signaling. The wt DVL1 plasmid significantly activated both reporters. In comparison both 1519ΔT and 1519* viruses did not activate the reporters as much as wt DVL1 . They did retain more activity than the control, parent plasmid. Statistical analysis done with one-way ANOVA followed by Dunnett’s multiple comparison test ( M ) or Tukey’s post-hoc test (C,D,N,O,P,Q). Scale bar in E-H = 2 mm, I-L = 20µm.
    Figure Legend Snippet: A) Mesenchymal cells harvested from the frontonasal mass of stage 24 (E4.5) embryos and were plated into high-density cultures. B) Other cultures were sectioned and used for microscopic analysis. C,E-H) There is a significant decrease in the proportion of cartilage from the DVL1 1519ΔT variant compared to the wtDVL1 infected cultures as measured in wholemount stained cultures. When the DVL1 1519* truncation was compared to wtDVL1 there was no significant difference in the Alcian blue stained area. The DVL1 1519* construct reduced cartilage compared to GFP controls. D,I-L) The cultures were significantly thinner in the presence of the DVL1 1519ΔT variant compared to w tDVL1 or the GFP controls. The DVL1 1519 * construct slightly reduced the thickness of the cartilage compared to GFP controls. M) The viruses containing human DVL1 constructs were expressed at similar levels in primary mesenchyme as determined by qRT-PCR with human-specific DVL1 primers. Generally the human DVL1 gene expression was elevated 10 to 15-fold by the viral transgenesis. N, O) The 1519ΔT virus significantly reduced expression of TWIST2 , MMP13 and LEF1 . P,Q) Two reporters were used in micromass cultures from frontonasal mass cells, the SuperTOPFlash reporter for canonical WNT signaling and ATF2 for JNK-PCP, non-canonical WNT signaling. The wt DVL1 plasmid significantly activated both reporters. In comparison both 1519ΔT and 1519* viruses did not activate the reporters as much as wt DVL1 . They did retain more activity than the control, parent plasmid. Statistical analysis done with one-way ANOVA followed by Dunnett’s multiple comparison test ( M ) or Tukey’s post-hoc test (C,D,N,O,P,Q). Scale bar in E-H = 2 mm, I-L = 20µm.

    Techniques Used: Variant Assay, Infection, Staining, Construct, Quantitative RT-PCR, Gene Expression, Virus, Expressing, Plasmid Preparation, Comparison, Activity Assay, Control

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    Luciferase:

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    Article Title: The abnormal C-terminus in DVL1 impacts Robinow Syndrome phenotypes
    Article Snippet: Micromass cultures were allowed to attach for 45 mins after plating and transfection reagents were added to the culture spot 30 mins prior to flooding the culture plate with micromass media. .. Firefly reporter plasmids: SuperTOPFlash (STF; 0.2ug, Addgene plasmid #12456) and Activating Transcription Factor 2 (0.4ug; ATF2) ( ) along with Renilla luciferase was transfected for normalization (0.01μg). .. Firefly reporter plasmids: SuperTOPFlash (STF; 0.2ug, Addgene plasmid #12456) and Activating Transcription Factor 2 (0.4ug; ATF2) ( ) along with Renilla luciferase was transfected for normalization (0.01μg).

    Construct:

    Article Title: TNIK inhibition abrogates colorectal cancer stemness.
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    Activity Assay:

    Article Title: TNIK inhibition abrogates colorectal cancer stemness.
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    Transfection:

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    Article Title: Lithium rescues synaptic plasticity and memory in Down syndrome mice
    Article Snippet: The cells were transfected through electroporation using an Amaxa nucleofector device (Lonza). .. For the luciferase experiments, the cells were transfected with SuperTOPFlash (in which Firefly luciferase expression is driven through multiple TCF-binding sites upstream of a minimal TATA box; Addgene plasmid 12456) or SuperFOPFlash (in which the TCF-binding sites are mutated; Addgene plasmid 12457) plasmids (60), together with the pRL-TK vector expressing Renilla luciferase to normalize for transfection efficiency. .. Twenty-four hours after transfection, the ADNPCs were treated with LiCl (2 mM) or the corresponding vehicle (2 mM NaCl) or recombinant Wnt3a (10 ng/ml; PeproTech), and the luciferase activity was measured at 24 hours later using a Dual-Luciferase Assay Kit (Promega) according to the manufacturer’s instructions.

    Article Title: DVL1 variants and C-terminal deletions have differential effects on craniofacial development and WNT signaling
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    Article Title: The abnormal C-terminus in DVL1 impacts Robinow Syndrome phenotypes
    Article Snippet: Micromass cultures were allowed to attach for 45 mins after plating and transfection reagents were added to the culture spot 30 mins prior to flooding the culture plate with micromass media. .. Firefly reporter plasmids: SuperTOPFlash (STF; 0.2ug, Addgene plasmid #12456) and Activating Transcription Factor 2 (0.4ug; ATF2) ( ) along with Renilla luciferase was transfected for normalization (0.01μg). .. Firefly reporter plasmids: SuperTOPFlash (STF; 0.2ug, Addgene plasmid #12456) and Activating Transcription Factor 2 (0.4ug; ATF2) ( ) along with Renilla luciferase was transfected for normalization (0.01μg).

    Expressing:

    Article Title: Lithium rescues synaptic plasticity and memory in Down syndrome mice
    Article Snippet: The cells were transfected through electroporation using an Amaxa nucleofector device (Lonza). .. For the luciferase experiments, the cells were transfected with SuperTOPFlash (in which Firefly luciferase expression is driven through multiple TCF-binding sites upstream of a minimal TATA box; Addgene plasmid 12456) or SuperFOPFlash (in which the TCF-binding sites are mutated; Addgene plasmid 12457) plasmids ( 60 ), together with the pRL-TK vector expressing Renilla luciferase to normalize for transfection efficiency. .. Twenty-four hours after transfection, the ADNPCs were treated with LiCl (2 mM) or the corresponding vehicle (2 mM NaCl) or recombinant Wnt3a (10 ng/ml; PeproTech), and the luciferase activity was measured at 24 hours later using a Dual-Luciferase Assay Kit (Promega) according to the manufacturer’s instructions.

    Article Title: Lithium rescues synaptic plasticity and memory in Down syndrome mice
    Article Snippet: The cells were transfected through electroporation using an Amaxa nucleofector device (Lonza). .. For the luciferase experiments, the cells were transfected with SuperTOPFlash (in which Firefly luciferase expression is driven through multiple TCF-binding sites upstream of a minimal TATA box; Addgene plasmid 12456) or SuperFOPFlash (in which the TCF-binding sites are mutated; Addgene plasmid 12457) plasmids (60), together with the pRL-TK vector expressing Renilla luciferase to normalize for transfection efficiency. .. Twenty-four hours after transfection, the ADNPCs were treated with LiCl (2 mM) or the corresponding vehicle (2 mM NaCl) or recombinant Wnt3a (10 ng/ml; PeproTech), and the luciferase activity was measured at 24 hours later using a Dual-Luciferase Assay Kit (Promega) according to the manufacturer’s instructions.

    Plasmid Preparation:

    Article Title: Lithium rescues synaptic plasticity and memory in Down syndrome mice
    Article Snippet: The cells were transfected through electroporation using an Amaxa nucleofector device (Lonza). .. For the luciferase experiments, the cells were transfected with SuperTOPFlash (in which Firefly luciferase expression is driven through multiple TCF-binding sites upstream of a minimal TATA box; Addgene plasmid 12456) or SuperFOPFlash (in which the TCF-binding sites are mutated; Addgene plasmid 12457) plasmids ( 60 ), together with the pRL-TK vector expressing Renilla luciferase to normalize for transfection efficiency. .. Twenty-four hours after transfection, the ADNPCs were treated with LiCl (2 mM) or the corresponding vehicle (2 mM NaCl) or recombinant Wnt3a (10 ng/ml; PeproTech), and the luciferase activity was measured at 24 hours later using a Dual-Luciferase Assay Kit (Promega) according to the manufacturer’s instructions.

    Article Title: Renal developmental defects resulting from in utero hypoxia are associated with suppression of ureteric β-catenin signaling.
    Article Snippet: Cell culture and luciferase assays UB2 cells49 cells were maintained in Dulbecco’s modified Eagle’s medium, 1 penicillin/streptomycin, 1 Glutamax I, 10% fetal calf serum (Life Technologies). .. For Luciferase assays, SuperTOPFlash or FOPFlash (Addgene Plasmid 12456 and 12457 respectively, Cambridge, MA)50 were transfected using Fugene HD (Promega, Alexandria, NSW, Australia) into UB2 cells seeded at 80% confluence. ..

    Article Title: Lithium rescues synaptic plasticity and memory in Down syndrome mice
    Article Snippet: The cells were transfected through electroporation using an Amaxa nucleofector device (Lonza). .. For the luciferase experiments, the cells were transfected with SuperTOPFlash (in which Firefly luciferase expression is driven through multiple TCF-binding sites upstream of a minimal TATA box; Addgene plasmid 12456) or SuperFOPFlash (in which the TCF-binding sites are mutated; Addgene plasmid 12457) plasmids (60), together with the pRL-TK vector expressing Renilla luciferase to normalize for transfection efficiency. .. Twenty-four hours after transfection, the ADNPCs were treated with LiCl (2 mM) or the corresponding vehicle (2 mM NaCl) or recombinant Wnt3a (10 ng/ml; PeproTech), and the luciferase activity was measured at 24 hours later using a Dual-Luciferase Assay Kit (Promega) according to the manufacturer’s instructions.

    Article Title: DVL1 variants and C-terminal deletions have differential effects on craniofacial development and WNT signaling
    Article Snippet: .. Firefly reporter plasmids: SuperTopflash (STF; 0.2ug, Addgene plasmid #12456) and Activating Transcription Factor 2 (0.4ug; ATF2) ( ) along with Renilla luciferase was transfected for normalization (0.01μg). ..

    Article Title: The abnormal C-terminus in DVL1 impacts Robinow Syndrome phenotypes
    Article Snippet: Micromass cultures were allowed to attach for 45 mins after plating and transfection reagents were added to the culture spot 30 mins prior to flooding the culture plate with micromass media. .. Firefly reporter plasmids: SuperTOPFlash (STF; 0.2ug, Addgene plasmid #12456) and Activating Transcription Factor 2 (0.4ug; ATF2) ( ) along with Renilla luciferase was transfected for normalization (0.01μg). .. Firefly reporter plasmids: SuperTOPFlash (STF; 0.2ug, Addgene plasmid #12456) and Activating Transcription Factor 2 (0.4ug; ATF2) ( ) along with Renilla luciferase was transfected for normalization (0.01μg).

    other:

    Article Title: Lithium rescues synaptic plasticity and memory in Down syndrome mice
    Article Snippet: The sections were counterstained with the nuclear dye Hoechst-33342 (Sigma-Aldrich).

    Article Title: Functional genomics in chicken embryos reveal the pathogenicity of two missense FZD2 variants associated with dominant Robinow syndrome
    Article Snippet: Micromass cultures were allowed to attach for 45 mins after plating and transfection reagents were added to the culture spot 30 mins prior to flooding the culture plate with micromass media.



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    Addgene inc 8x supertopflash addgene
    (A): HEK293T cells were transiently transfected with firefly reporter plasmids for <t>SuperTopFlash</t> together with a fixed amount of each GFP tagged DVL1 construct or with a 1:1 stoichiometric ratio of two DVL1 constructs with the same total amount of DVL1 constructs. Data represents average values of relative TOPFlash over FOPFlash ratios from one representative experiment performed in triplicate. (B): HEK293T cells were transiently transfected with firefly reporter plasmids for SuperTopFlash together with a fixed amount of each FLAG tagged DVL2 construct or with a 1:1 stoichiometric ratio of two DVL2 constructs with the same total amount of DVL2 constructs. Data represents average values of relative TOPFlash over FOPFlash ratios from one representative experiment performed in triplicate. (C): HEK293T cells were transiently transfected with firefly reporter plasmids for SuperTopFlash together with a fixed amount of each HA tagged DVL3 construct or with a 1:1 stoichiometric ratio of two DVL3 constructs with the same total amount of DVL3 constructs. Data represents average values of relative TOPFlash over FOPFlash ratios from one representative experiment performed in triplicate. Individual p values were calculated with student t-tests and denote the difference between DVL-WT and the other conditions without WNT stimulation, difference between each condition with or without stimulation was also calculated. An asterisk indicates a significant difference: *: p < 0.05; **: p < 0.005; ***: p < 0.0005; ****: p < 0.0001.
    8x Supertopflash Addgene, supplied by Addgene inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/supertopflash/M50+Super+8x+TOPFlash+(Plasmid+%2312456)/pmc12103576__41467_2025_60096_MOESM1_ESM-85-45-47
    Average 96 stars, based on 1 article reviews
    8x supertopflash addgene - by Bioz Stars, 2026-11
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    hek293 supertopflash  (ATCC)
    95
    ATCC hek293 supertopflash
    (A): HEK293T cells were transiently transfected with firefly reporter plasmids for <t>SuperTopFlash</t> together with a fixed amount of each GFP tagged DVL1 construct or with a 1:1 stoichiometric ratio of two DVL1 constructs with the same total amount of DVL1 constructs. Data represents average values of relative TOPFlash over FOPFlash ratios from one representative experiment performed in triplicate. (B): HEK293T cells were transiently transfected with firefly reporter plasmids for SuperTopFlash together with a fixed amount of each FLAG tagged DVL2 construct or with a 1:1 stoichiometric ratio of two DVL2 constructs with the same total amount of DVL2 constructs. Data represents average values of relative TOPFlash over FOPFlash ratios from one representative experiment performed in triplicate. (C): HEK293T cells were transiently transfected with firefly reporter plasmids for SuperTopFlash together with a fixed amount of each HA tagged DVL3 construct or with a 1:1 stoichiometric ratio of two DVL3 constructs with the same total amount of DVL3 constructs. Data represents average values of relative TOPFlash over FOPFlash ratios from one representative experiment performed in triplicate. Individual p values were calculated with student t-tests and denote the difference between DVL-WT and the other conditions without WNT stimulation, difference between each condition with or without stimulation was also calculated. An asterisk indicates a significant difference: *: p < 0.05; **: p < 0.005; ***: p < 0.0005; ****: p < 0.0001.
    Hek293 Supertopflash, supplied by ATCC, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    hek293 supertopflash stf  (ATCC)
    95
    ATCC hek293 supertopflash stf
    Poly(A) tail mimetics enhance the expression of target mRNAs (A) Schematic of the basic design of the mRNA booster: a 30-nucleotide sequence complementary to a particular region of the 3′ UTR of a targeted mRNA, which bears a poly(A) tail. (B) A poly(A) mimetic enhances the expression of an in vitro transcribed Firefly luciferase reporter in <t>HEK293</t> cells, with a significant (one-way ANOVA, ∗∗∗∗ p < 0.00001) 6-fold increase in FLuc over Renilla activity when the booster bears a 200-nucleotide poly(A) tail, 48 h after transfection. The results are depicted for a booster without a poly(A) tail, with a short 10-nucleotide tail, or a long 200-nucleotide tail; for two different FLuc mRNA to booster ratios (low dose, 1:36; and high dose, 1:216). (C) Targeting an endogenous mRNA confirms the booster efficiency to enhance cellular gene expression. LSM8 mRNA levels normalized to GAPDH are represented, after treatment with 20 nM final concentration of LSM8- specific booster compared to a non-specific polyadenylated control. Cells were harvested 2, 4, 6, 8, 16, and 24 h after transfection. The comparison between control vs. booster treated in each time point indicates significant increase (∗∗) after 4-, 16- and 24-h incubation. Two-way ANOVA, ∗∗ p < 0.001, ∗∗∗∗ p < 0.00001.
    Hek293 Supertopflash Stf, supplied by ATCC, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/supertopflash/HEK+293+STF/pmc11834087-240-11-16
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    Image Search Results


    A) Mesenchymal cells harvested from the frontonasal mass of stage 24 (E4.5) embryos and were plated into high-density cultures. B) Other cultures were sectioned and used for microscopic analysis. C,E-H) There is a significant decrease in the proportion of cartilage from the DVL1 1519ΔT variant compared to the wtDVL1 infected cultures as measured in wholemount stained cultures. When the DVL1 1519* truncation was compared to wtDVL1 there was no significant difference in the Alcian blue stained area. The DVL1 1519* construct reduced cartilage compared to GFP controls. D,I-L) The cultures were significantly thinner in the presence of the DVL1 1519ΔT variant compared to w tDVL1 or the GFP controls. The DVL1 1519 * construct slightly reduced the thickness of the cartilage compared to GFP controls. M) The viruses containing human DVL1 constructs were expressed at similar levels in primary mesenchyme as determined by qRT-PCR with human-specific DVL1 primers. Generally the human DVL1 gene expression was elevated 10 to 15-fold by the viral transgenesis. N, O) The 1519ΔT virus significantly reduced expression of TWIST2 , MMP13 and LEF1 . P,Q) Two reporters were used in micromass cultures from frontonasal mass cells, the SuperTOPFlash reporter for canonical WNT signaling and ATF2 for JNK-PCP, non-canonical WNT signaling. The wt DVL1 plasmid significantly activated both reporters. In comparison both 1519ΔT and 1519* viruses did not activate the reporters as much as wt DVL1 . They did retain more activity than the control, parent plasmid. Statistical analysis done with one-way ANOVA followed by Dunnett’s multiple comparison test ( M ) or Tukey’s post-hoc test (C,D,N,O,P,Q). Scale bar in E-H = 2 mm, I-L = 20µm.

    Journal: bioRxiv

    Article Title: The abnormal C-terminus in DVL1 impacts Robinow Syndrome phenotypes

    doi: 10.64898/2026.02.14.705933

    Figure Lengend Snippet: A) Mesenchymal cells harvested from the frontonasal mass of stage 24 (E4.5) embryos and were plated into high-density cultures. B) Other cultures were sectioned and used for microscopic analysis. C,E-H) There is a significant decrease in the proportion of cartilage from the DVL1 1519ΔT variant compared to the wtDVL1 infected cultures as measured in wholemount stained cultures. When the DVL1 1519* truncation was compared to wtDVL1 there was no significant difference in the Alcian blue stained area. The DVL1 1519* construct reduced cartilage compared to GFP controls. D,I-L) The cultures were significantly thinner in the presence of the DVL1 1519ΔT variant compared to w tDVL1 or the GFP controls. The DVL1 1519 * construct slightly reduced the thickness of the cartilage compared to GFP controls. M) The viruses containing human DVL1 constructs were expressed at similar levels in primary mesenchyme as determined by qRT-PCR with human-specific DVL1 primers. Generally the human DVL1 gene expression was elevated 10 to 15-fold by the viral transgenesis. N, O) The 1519ΔT virus significantly reduced expression of TWIST2 , MMP13 and LEF1 . P,Q) Two reporters were used in micromass cultures from frontonasal mass cells, the SuperTOPFlash reporter for canonical WNT signaling and ATF2 for JNK-PCP, non-canonical WNT signaling. The wt DVL1 plasmid significantly activated both reporters. In comparison both 1519ΔT and 1519* viruses did not activate the reporters as much as wt DVL1 . They did retain more activity than the control, parent plasmid. Statistical analysis done with one-way ANOVA followed by Dunnett’s multiple comparison test ( M ) or Tukey’s post-hoc test (C,D,N,O,P,Q). Scale bar in E-H = 2 mm, I-L = 20µm.

    Article Snippet: Firefly reporter plasmids: SuperTOPFlash (STF; 0.2ug, Addgene plasmid #12456) and Activating Transcription Factor 2 (0.4ug; ATF2) ( ) along with Renilla luciferase was transfected for normalization (0.01μg).

    Techniques: Variant Assay, Infection, Staining, Construct, Quantitative RT-PCR, Gene Expression, Virus, Expressing, Plasmid Preparation, Comparison, Activity Assay, Control

    (A): HEK293T cells were transiently transfected with firefly reporter plasmids for SuperTopFlash together with a fixed amount of each GFP tagged DVL1 construct or with a 1:1 stoichiometric ratio of two DVL1 constructs with the same total amount of DVL1 constructs. Data represents average values of relative TOPFlash over FOPFlash ratios from one representative experiment performed in triplicate. (B): HEK293T cells were transiently transfected with firefly reporter plasmids for SuperTopFlash together with a fixed amount of each FLAG tagged DVL2 construct or with a 1:1 stoichiometric ratio of two DVL2 constructs with the same total amount of DVL2 constructs. Data represents average values of relative TOPFlash over FOPFlash ratios from one representative experiment performed in triplicate. (C): HEK293T cells were transiently transfected with firefly reporter plasmids for SuperTopFlash together with a fixed amount of each HA tagged DVL3 construct or with a 1:1 stoichiometric ratio of two DVL3 constructs with the same total amount of DVL3 constructs. Data represents average values of relative TOPFlash over FOPFlash ratios from one representative experiment performed in triplicate. Individual p values were calculated with student t-tests and denote the difference between DVL-WT and the other conditions without WNT stimulation, difference between each condition with or without stimulation was also calculated. An asterisk indicates a significant difference: *: p < 0.05; **: p < 0.005; ***: p < 0.0005; ****: p < 0.0001.

    Journal: bioRxiv

    Article Title: Pathogenic DVL frameshifting variants in Robinow syndrome disrupt WNT signaling and cellular dynamics

    doi: 10.1101/2025.08.02.668297

    Figure Lengend Snippet: (A): HEK293T cells were transiently transfected with firefly reporter plasmids for SuperTopFlash together with a fixed amount of each GFP tagged DVL1 construct or with a 1:1 stoichiometric ratio of two DVL1 constructs with the same total amount of DVL1 constructs. Data represents average values of relative TOPFlash over FOPFlash ratios from one representative experiment performed in triplicate. (B): HEK293T cells were transiently transfected with firefly reporter plasmids for SuperTopFlash together with a fixed amount of each FLAG tagged DVL2 construct or with a 1:1 stoichiometric ratio of two DVL2 constructs with the same total amount of DVL2 constructs. Data represents average values of relative TOPFlash over FOPFlash ratios from one representative experiment performed in triplicate. (C): HEK293T cells were transiently transfected with firefly reporter plasmids for SuperTopFlash together with a fixed amount of each HA tagged DVL3 construct or with a 1:1 stoichiometric ratio of two DVL3 constructs with the same total amount of DVL3 constructs. Data represents average values of relative TOPFlash over FOPFlash ratios from one representative experiment performed in triplicate. Individual p values were calculated with student t-tests and denote the difference between DVL-WT and the other conditions without WNT stimulation, difference between each condition with or without stimulation was also calculated. An asterisk indicates a significant difference: *: p < 0.05; **: p < 0.005; ***: p < 0.0005; ****: p < 0.0001.

    Article Snippet: Cells were transfected using Lipofectamine 3000 (Invitrogen, Cat. No. L3000-008) with plasmids containing the wild type or variant DVL genes plus firefly reporter plasmids for SuperTopFlash (M50 Super 8x TOPFlash, Addgene plasmid #12456) and FOPFlash (M51 Super 8x FOPFlash, Addgene plasmid #12457) plasmid used as a negative control for “leaky” firefly luciferase transcription.

    Techniques: Transfection, Construct

    Poly(A) tail mimetics enhance the expression of target mRNAs (A) Schematic of the basic design of the mRNA booster: a 30-nucleotide sequence complementary to a particular region of the 3′ UTR of a targeted mRNA, which bears a poly(A) tail. (B) A poly(A) mimetic enhances the expression of an in vitro transcribed Firefly luciferase reporter in HEK293 cells, with a significant (one-way ANOVA, ∗∗∗∗ p < 0.00001) 6-fold increase in FLuc over Renilla activity when the booster bears a 200-nucleotide poly(A) tail, 48 h after transfection. The results are depicted for a booster without a poly(A) tail, with a short 10-nucleotide tail, or a long 200-nucleotide tail; for two different FLuc mRNA to booster ratios (low dose, 1:36; and high dose, 1:216). (C) Targeting an endogenous mRNA confirms the booster efficiency to enhance cellular gene expression. LSM8 mRNA levels normalized to GAPDH are represented, after treatment with 20 nM final concentration of LSM8- specific booster compared to a non-specific polyadenylated control. Cells were harvested 2, 4, 6, 8, 16, and 24 h after transfection. The comparison between control vs. booster treated in each time point indicates significant increase (∗∗) after 4-, 16- and 24-h incubation. Two-way ANOVA, ∗∗ p < 0.001, ∗∗∗∗ p < 0.00001.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Use of polyadenosine tail mimetics to enhance mRNA expression from genes associated with haploinsufficiency disorders

    doi: 10.1016/j.omtn.2025.102453

    Figure Lengend Snippet: Poly(A) tail mimetics enhance the expression of target mRNAs (A) Schematic of the basic design of the mRNA booster: a 30-nucleotide sequence complementary to a particular region of the 3′ UTR of a targeted mRNA, which bears a poly(A) tail. (B) A poly(A) mimetic enhances the expression of an in vitro transcribed Firefly luciferase reporter in HEK293 cells, with a significant (one-way ANOVA, ∗∗∗∗ p < 0.00001) 6-fold increase in FLuc over Renilla activity when the booster bears a 200-nucleotide poly(A) tail, 48 h after transfection. The results are depicted for a booster without a poly(A) tail, with a short 10-nucleotide tail, or a long 200-nucleotide tail; for two different FLuc mRNA to booster ratios (low dose, 1:36; and high dose, 1:216). (C) Targeting an endogenous mRNA confirms the booster efficiency to enhance cellular gene expression. LSM8 mRNA levels normalized to GAPDH are represented, after treatment with 20 nM final concentration of LSM8- specific booster compared to a non-specific polyadenylated control. Cells were harvested 2, 4, 6, 8, 16, and 24 h after transfection. The comparison between control vs. booster treated in each time point indicates significant increase (∗∗) after 4-, 16- and 24-h incubation. Two-way ANOVA, ∗∗ p < 0.001, ∗∗∗∗ p < 0.00001.

    Article Snippet: We tested our mRNA booster technology in HEK293 (ATCC CRL 1573), HEK293 SuperTopFlash (STF), and SH-SY5Y (ATCC CRL 2266) cell lines as well as iPSC-derived neuronal cells (see below).

    Techniques: Expressing, Sequencing, In Vitro, Luciferase, Activity Assay, Transfection, Gene Expression, Concentration Assay, Control, Comparison, Incubation

    mRNA boosters enhance haploinsufficiency-associated mRNAs (A) Schematic of the guide sequences position on the 3′ UTR of their target genes: MECP2 , CTNNB1 , PURA , and SYNGAP1 mRNAs, used throughout the study. Guide sequences spanning distinct regions were evaluated and assigned. (B) Levels of M E CP2 mRNA measured by qRT-PCR in SH-SY5Y cells transfected with 40 nM booster V.2.2 (MB2) against 3′ UTR of the M E CP2 or a control booster not targeting M E CP2 (control) and harvested 16 h after transfection. Welch’s t test, ∗ p = 0.05. (C and D) Levels of CTNNB1 (C) and PURA (D) mRNAs measured by qRT-PCR in HEK293 cells transfected with boosters V.2.2 targeting distinct regions of the 3′ UTR (B1 and B2) or a non-specific control (control), 24 h after transfection. Ordinary one-way ANOVA, ∗ p = 0.05, ∗∗ p = 0.005. (E) S YN GAP1 mRNA levels measured by qRT-PCR in SH-SY5Y cells transfected with two versions of booster V.2.0 (SB1 and SB2) targeting S YN GAP1 mRNA 3′ UTR or a non-specific control (control). SB2 showed a significant (∗) up to 4-fold increase in the mRNA level. Welch’s t test, ∗ p = 0.05.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Use of polyadenosine tail mimetics to enhance mRNA expression from genes associated with haploinsufficiency disorders

    doi: 10.1016/j.omtn.2025.102453

    Figure Lengend Snippet: mRNA boosters enhance haploinsufficiency-associated mRNAs (A) Schematic of the guide sequences position on the 3′ UTR of their target genes: MECP2 , CTNNB1 , PURA , and SYNGAP1 mRNAs, used throughout the study. Guide sequences spanning distinct regions were evaluated and assigned. (B) Levels of M E CP2 mRNA measured by qRT-PCR in SH-SY5Y cells transfected with 40 nM booster V.2.2 (MB2) against 3′ UTR of the M E CP2 or a control booster not targeting M E CP2 (control) and harvested 16 h after transfection. Welch’s t test, ∗ p = 0.05. (C and D) Levels of CTNNB1 (C) and PURA (D) mRNAs measured by qRT-PCR in HEK293 cells transfected with boosters V.2.2 targeting distinct regions of the 3′ UTR (B1 and B2) or a non-specific control (control), 24 h after transfection. Ordinary one-way ANOVA, ∗ p = 0.05, ∗∗ p = 0.005. (E) S YN GAP1 mRNA levels measured by qRT-PCR in SH-SY5Y cells transfected with two versions of booster V.2.0 (SB1 and SB2) targeting S YN GAP1 mRNA 3′ UTR or a non-specific control (control). SB2 showed a significant (∗) up to 4-fold increase in the mRNA level. Welch’s t test, ∗ p = 0.05.

    Article Snippet: We tested our mRNA booster technology in HEK293 (ATCC CRL 1573), HEK293 SuperTopFlash (STF), and SH-SY5Y (ATCC CRL 2266) cell lines as well as iPSC-derived neuronal cells (see below).

    Techniques: Quantitative RT-PCR, Transfection, Control

    Poly(A)-tail mimetics increase CTNNB1 expression in different cell types (A) Schematic of five distinct boosters along CTNNB1 3′ UTR, designed between nucleotide 376 and 1024. (B) The bar graph presents the result of quantified western blotting ( <xref ref-type=Figure S3 ) for CTNNB1 , showing the position and dose-dependent efficiency of the boosters targeting CTNNB1 compared to a non-specific booster control. To screen and select the most effective booster for CTNNB1 , HEK293-STF cells were transfected with different doses (10, 20, 30, 40, 50, and 100 nM) of five distinct CTNNB1 boosters V.1.0 (CB3, CB4, CB5, CB6, and CB7). Booster CB7 increases the protein level up to 2-fold compared to control at 40 nM booster or higher. (C) Representative western blotting and quantifications for three biological replicates showing the significant dose-dependent increase of CTNNB1 protein levels upon exposure to booster CB7 compared to control (for more blots and loading control, see Figure S3 ). Two-way ANOVA, ∗ p = 0.05, ∗∗∗ p = 0.0005. (D) qRT-PCR analysis confirms booster CB7 efficiency to increase CTNNB1 mRNA levels in a dose-dependent manner. Two-way ANOVA, ∗ p = 0.01. (E and F) mRNA levels of Wnt signaling pathway markers measured by qRT-PCR, illustrating the functional enhancement of CTNNB1 following the increase in CTNNB1 expression, in two different cell lines: HEK293-STF (E) and SH-SY5Y (F) cells. Welch’s t test, ∗ p < 0.05. (G and H) Protein levels of B-catenin and its downstream effector EN2 in β-catenin heterozygote, human iPSC-derived neurons, 48 h after transfection with LNP-packed boosters against CTNNB1 (CB7) or a non-specific control. Three different LNP formulations were tested (LNP1, LNP2, and LNP10; see Figure S2 ). (G) A representative western blot and (H) a quantitation of CTNNB1 and EN2 from western blots from two biological replicates. " width="100%" height="100%">

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Use of polyadenosine tail mimetics to enhance mRNA expression from genes associated with haploinsufficiency disorders

    doi: 10.1016/j.omtn.2025.102453

    Figure Lengend Snippet: Poly(A)-tail mimetics increase CTNNB1 expression in different cell types (A) Schematic of five distinct boosters along CTNNB1 3′ UTR, designed between nucleotide 376 and 1024. (B) The bar graph presents the result of quantified western blotting ( Figure S3 ) for CTNNB1 , showing the position and dose-dependent efficiency of the boosters targeting CTNNB1 compared to a non-specific booster control. To screen and select the most effective booster for CTNNB1 , HEK293-STF cells were transfected with different doses (10, 20, 30, 40, 50, and 100 nM) of five distinct CTNNB1 boosters V.1.0 (CB3, CB4, CB5, CB6, and CB7). Booster CB7 increases the protein level up to 2-fold compared to control at 40 nM booster or higher. (C) Representative western blotting and quantifications for three biological replicates showing the significant dose-dependent increase of CTNNB1 protein levels upon exposure to booster CB7 compared to control (for more blots and loading control, see Figure S3 ). Two-way ANOVA, ∗ p = 0.05, ∗∗∗ p = 0.0005. (D) qRT-PCR analysis confirms booster CB7 efficiency to increase CTNNB1 mRNA levels in a dose-dependent manner. Two-way ANOVA, ∗ p = 0.01. (E and F) mRNA levels of Wnt signaling pathway markers measured by qRT-PCR, illustrating the functional enhancement of CTNNB1 following the increase in CTNNB1 expression, in two different cell lines: HEK293-STF (E) and SH-SY5Y (F) cells. Welch’s t test, ∗ p < 0.05. (G and H) Protein levels of B-catenin and its downstream effector EN2 in β-catenin heterozygote, human iPSC-derived neurons, 48 h after transfection with LNP-packed boosters against CTNNB1 (CB7) or a non-specific control. Three different LNP formulations were tested (LNP1, LNP2, and LNP10; see Figure S2 ). (G) A representative western blot and (H) a quantitation of CTNNB1 and EN2 from western blots from two biological replicates.

    Article Snippet: We tested our mRNA booster technology in HEK293 (ATCC CRL 1573), HEK293 SuperTopFlash (STF), and SH-SY5Y (ATCC CRL 2266) cell lines as well as iPSC-derived neuronal cells (see below).

    Techniques: Expressing, Western Blot, Control, Transfection, Quantitative RT-PCR, Functional Assay, Derivative Assay, Quantitation Assay