supertopflash  (Addgene inc)

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

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