Journal: The Journal of Biological Chemistry

Article Title: Pathogenic variants in KCTD1 disrupt cAMP signaling and cellular communication associated with developmental pathways

doi: 10.1016/j.jbc.2025.110813

Figure Lengend Snippet: Comparison of KCTD1 mutations on Wnt, Hh, and Notch signaling. A , schematic of 3P-Luc triple reporter luminescence assay. A single construct enables monitoring of Wnt (Nluc), Hh (Fluc), and Notch (Gluc) pathways. Wnt signaling was stimulated by treatment with Wnt3a peptide. Hh signaling was stimulated by cotransfection with Gli1. Notch signaling was stimulated by cotransfection with ICN1-GFP. B , luminescence quantification (Nano-Glo; Nluc) to HEK293 cells cotransfected with 3P-Luc, KCTD1, and pcDNA following overnight treatment with 100 ng/ml Wnt3a. n = 3 to 4 independent experiments. C , 3P-Luc Wnt response quantification in HEK293 cells cotransfected with KCTD1 variants (1.0 μg). n = 3 independent experiments. D , luminescence quantification (Bright-Glo; Fluc) to HEK293 cells cotransfected with 3P-Luc, Gli1, KCTD1, and pcDNA. n = 3 to 4 independent experiments. E , 3P-Luc Hh response quantification in HEK293 cells cotransfected with KCTD1 variants (1.0 μg). n = 3 independent experiments. F , luminescence quantification (Coelenterazine; Gluc) to HEK293 cells cotransfected with 3P-Luc, ICN1-GFP, KCTD1, and pcDNA. n = 4 independent experiments. G , 3P-Luc Notch response quantification in HEK293 cells cotransfected with KCTD1 variants (1.0 μg). n = 3 independent experiments. One-way ANOVA, Dunnett posttest, multiple comparison to pcDNA group; ∗ p < 0.05, ∗∗∗∗ p < 0.0001. 3P-Luc, triple reporter plasmid; Fluc, Firefly luciferase; Gluc, Gaussia luciferase; Hh, Hedgehog; KCTD1, potassium channel tetramerization domain-containing 1; Nluc, Nanoluciferase.

Article Snippet: ICN1-mVenus was generated by insertion of synthesized ICN1 ORF (Twist Bioscience) at the N terminus of the mVenus-N1 vector using KpnI and AgeI sites.

Techniques: Comparison, Luminescence Assay, Construct, Cotransfection, Plasmid Preparation, Luciferase

Journal: The Journal of Biological Chemistry

Article Title: Pathogenic variants in KCTD1 disrupt cAMP signaling and cellular communication associated with developmental pathways

doi: 10.1016/j.jbc.2025.110813

Figure Lengend Snippet: KCTD1 interacts with RBPJ and regulates Notch signaling. A , schematic for IP luminescence strategy for HEK293 cells cotransfected with Fluc-KCTD1-flag and Nluc-tagged proteins of interest (POI) in the Notch signaling cascade. B , quantification of Nluc luminescence ratio (IP/total lysate) from HEK293 cells cotransfected with Fluc-KCTD1-flag and Nluc-tagged POI. n = 4 independent experiments. C , IP of KCTD1-mVenus from HEK293 cells with an anti-GFP antibody followed by Western blot for HA-RBPJ with an anti-HA antibody. Representative blot from three independent experiments. D , Notch response quantification (Coelenterazine; Gluc) in HEK293 cotransfected with 3P-Luc and ICN1-GFP along with pcDNA, KCTD1-WT, and RBPJ as indicated. n = 5 independent experiments. One-way ANOVA, Dunnett posttest, and multiple comparison to pcDNA group; ∗∗∗∗ p < 0.0001. E , same data as panel 3C with omission of pcDNA group (n = 5 independent experiments). One-way ANOVA, Dunnett posttest, multiple comparison to KCTD1 group; ∗∗∗∗ p < 0.0001. Fluc, Firefly luciferase; KCTD1, potassium channel tetramerization domain-containing 1; KCTD1-WT, wildtype KCTD1; RBPJ, repressor recombination signal binding protein for immunoglobulin kappa J region.

Article Snippet: ICN1-mVenus was generated by insertion of synthesized ICN1 ORF (Twist Bioscience) at the N terminus of the mVenus-N1 vector using KpnI and AgeI sites.

Techniques: Western Blot, Comparison, Luciferase, Binding Assay

Journal: Scientific Reports

Article Title: Notch signaling regulates UNC5B to suppress endothelial proliferation, migration, junction activity, and retinal plexus branching

doi: 10.1038/s41598-024-64375-z

Figure Lengend Snippet: UNC5B is a novel target of Notch in endothelial cells. ( A , B ) Induction of Notch signaling in Human Umbilical Vein Endothelial Cells (HUVEC) using DLL4-Fc coated TLA plates significantly upregulate expression of Notch target genes HES1, HEY1, NRARP, and DLL4 (blue bars). This effect is blocked by GSI treatment with Compound E (CpE, pink bars). UNC5B expression is significantly upregulated by Notch signaling and this upregulation is significantly blocked by CpE (right). Expression levels were evaluated by qPCR. ( C – E ) EGTA induction (black lines) and CPE inhibition of EGTA (red lines) of canonical Notch target HEY1 ( C ), UNC5B ( D ), and GAPDH ( E ) in HUVECs. (F, G ) HUVECs were lentivirally transduced with control RFP, ICN1, or ICN4 expression constructs. 24 h after lentivirus infection, cells were harvested and analyzed by qPCR for NOTCH1 , NOTCH4 , and UNC5B expression. Two-way ANOVA ( A , B ), multiple unpaired t-tests ( C – E ) and one-way ANOVA ( F , G ), presented as mean ± s.e.m. from at least 3 different biological replicates per experiment.

Article Snippet: Cdh5-CreER T2 mice were also bred with Unc5B flox ( Unc5B tm1(flox)Slac/Slac ) mice and ICN1 floxstop ( Gt(ROSA)26So rtm1(Notch1) Dam/J , Jackson Labs strain #008159) mice to generate mice with endothelial cell (EC)-specific loss of Unc5B and EC-specific active Notch signaling.

Techniques: Expressing, Inhibition, Transduction, Control, Construct, Infection

Journal: Scientific Reports

Article Title: Notch signaling regulates UNC5B to suppress endothelial proliferation, migration, junction activity, and retinal plexus branching

doi: 10.1038/s41598-024-64375-z

Figure Lengend Snippet: UNC5B regulates endothelial cell proliferation and migration downstream of Notch signaling. HUVECs were lentivirally transduced with scramble control (shCNT) or shRNA targeting UNC5B (shUNC5B). ( A ) qPCR for UNC5B expression 24 h after lentivirus infection. (B) Representative images of shCNT and shUNC5B HUVEC cell morphology. Yellow boxed inserts show zoomed images for cellular morphology. ( C ) shCNT and shUNC5B HUVEC proliferation measured by MTT assay. ( D ) Percent closure of scratch wounds in shCNT and shUNC5B HUVEC monolayers. Asterisks indicate the time points for significant differences in migration between shCNT and shUNC5B. ( E ) Proliferation of PMVECs transduced with ICN1 or ICN4 expression vectors and shCNT or shUNC5B, relative to shCNT-RFP control. ( F ) Cell migration values of PMVECs transduced with ICN1 or ICN4 expression vectors and shCNT or shUNC5B, relative to shCNT-RFP control. Unpaired t-tests ( A , C ) and multiple comparison unpaired t-tests ( D – F ), presented as mean ± s.e.m. Each dot represents an independent experiment with 4 replicates per experiment. Scale bars, 150 μm and 35 μm (zoomed).

Article Snippet: Cdh5-CreER T2 mice were also bred with Unc5B flox ( Unc5B tm1(flox)Slac/Slac ) mice and ICN1 floxstop ( Gt(ROSA)26So rtm1(Notch1) Dam/J , Jackson Labs strain #008159) mice to generate mice with endothelial cell (EC)-specific loss of Unc5B and EC-specific active Notch signaling.

Techniques: Migration, Transduction, Control, shRNA, Expressing, Infection, MTT Assay, Comparison

Journal: Scientific Reports

Article Title: Notch signaling regulates UNC5B to suppress endothelial proliferation, migration, junction activity, and retinal plexus branching

doi: 10.1038/s41598-024-64375-z

Figure Lengend Snippet: Endothelial cell-specific Notch activation increases Unc5B expression. ( A ) Representative postnatal day (P) 5 retina distinguishing the immature vascular plexus of the angiogenic front region from the central region, which has increasingly mature arteries, capillaries, and veins. ( B ) P5 retina stained with anti-Unc5B (red) and Isolectin B4 (IB4, green) to label the blood vessels at the angiogenic front. ( C ) P5 retina stained with anti-Unc5B and VE-cadherin (VE-cad, green) to label the maturing vessels. A = artery and V = vein in all panels. ( D ) Diagram of tamoxifen administration to ICN1 IOE-EC mice and harvest at P5 for analysis. ( E ) Detection of ICN1 in endothelial nuclei of ICN1 IOE-EC retina with anti-GFP antibody. ( F ) ICN1 IOE-EC mutant and control mouse retinas stained for Unc5B (red) and vasculature (VE-cad, green; IB4, blue). ( F’ ) Isolated Unc5B channel depicted in grayscale. ( G ) Quantification of the percentage of Unc5B fluorescence intensity in arteries, capillaries and veins. At least 17 representative fields of view were examined and averaged from five distinct control mice and six distinct ICN1 OE-EC mice. Multiple comparisons unpaired t-test, presented as mean ± s.e.m. Scale bars, 230 μm ( A ), 100 μm ( B – E ), and 100 μm ( F ).

Article Snippet: Cdh5-CreER T2 mice were also bred with Unc5B flox ( Unc5B tm1(flox)Slac/Slac ) mice and ICN1 floxstop ( Gt(ROSA)26So rtm1(Notch1) Dam/J , Jackson Labs strain #008159) mice to generate mice with endothelial cell (EC)-specific loss of Unc5B and EC-specific active Notch signaling.

Techniques: Activation Assay, Expressing, Staining, Mutagenesis, Control, Isolation, Fluorescence