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

Journal: Molecular Oncology

Article Title: Characterization of the spectrum of trivalent VAV1 ‐mutation‐driven tumours using a gene‐edited mouse model

doi: 10.1002/1878-0261.13295

Figure Lengend Snippet: Expression of VAV1 ΔC promotes CD4 + T cell lymphomagenesis. (A, B) Representative view (A) and quantification of the weight (B) of spleen from mice of the indicated phenotypes at the time of euthanasia. In A, scale bar = 1 cm. In B, each dot represents a single experimental mouse. n = 12 animals per genotype, except in the case of Trp53 ER/ER ;Vav1 ΔC/ΔC mice ( n = 7). (C, D) Example of haematoxylin–eosin‐stained sections of the spleen (C) and lymph nodes (D) from animals of the indicated genotypes (top) at the time of euthanasia. In C, arrows indicate the presence of both megakaryocytes and leukemic cells. RP, red pulp; WP, white pulp. In D, C, cortex; M, medulla; V, venules. Scale bars, 10 (C, D, top panels) and 100 (C, D, bottom panels) μm. n = 5 independent tissue sections per genotype. (E) Representative flow cytometry plots showing the levels of surface expression of CD4 (top panels), PD1 and CXCR5 (middle panels) and ICOS plus CD69 (bottom panels) in splenocytes isolated from mice of indicated genotypes (top) at the time of euthanasia. n = 10 animals per genotype. In all cases, the numbers indicate the relative percentage (%) of the interrogated cell subpopulation in total (top raw of panels) or gated CD4 + T (middle raw of panels) and CD4 + T FH (bottom raw of panels) cells. n = 8 ( Trp53 ER/ER ), 9 ( Vav1 ΔC/ΔC ) and 7 ( Trp53 ER/ER ;Vav1 ΔC/ΔC ) mice. (F) Flow cytometry determination of the CD4 + versus CD8 + T cell ratio in spleen from the mice of the indicated genotypes (bottom) at the time of euthanasia. Each point represents the values obtained with a single experimental mouse. n as in E. (G) Quantification of the percentage of T FH cells in the population of CD4 + ‐gated splenocytes from mice of the indicated genotypes (bottom) at the time of euthanasia. Each point represents the values obtained with a single experimental mouse. n as in E. (H, I) Flow cytometry determination of the surface levels of ICOS (H) and CD69 (I) in CD4 + T FH ‐gated splenocytes from animals of indicated genotypes (bottom) at the time of euthanasia. f.i., mean fluorescence intensity relative to the isotype‐matched control antibody. In both panels, each point represents the values obtained with a single experimental mouse. n as in E. (J–L) Flow cytometry determination of p‐AKT (J), p‐ERK1/2 (K) and ICN1 (L) levels in CD4 + T FH ‐gated splenocytes from 8‐month‐old mice of the indicated genotypes at the time of euthanasia. n as in E. (M) qRT‐PCR analyses showing the expression of indicated ICN1 targets in lymphoma cell samples from the mouse models shown in the inset. n = 3. Data information: In panels B and F–M, the values represent the mean ± SEM. Statistical values are given relative to appropriate control animals. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001 (Chi‐squared test). [Colour figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: For intracellular ICN1 staining, cells were fixed with Cytofix/Cytoperm (Cat. No. 554714; BD Bioscience, Franklin Lakes, NJ, USA) for 10 min and stained with PE‐labelled antibodies to either TOX (Cat. No. 12‐6502‐82; eBiosciences; 1 : 50 dilution) or ICN1 (mN1A, Cat. No. 552768; Cell Signaling Technologies; 1 : 50 dilution) for 1 h at room temperature in phosphate‐buffered saline solution supplemented with 5% fetal bovine serum and 10% saponin.

Techniques: Expressing, Staining, Flow Cytometry, Isolation, Fluorescence, Quantitative RT-PCR

Journal: Molecular Oncology

Article Title: Characterization of the spectrum of trivalent VAV1 ‐mutation‐driven tumours using a gene‐edited mouse model

doi: 10.1002/1878-0261.13295

Figure Lengend Snippet: VAV1 ΔC favours progression of K‐RAS G12D ‐driven lung tumours. (A, B) Quantification of type of hyperplasia (A) and grade of tumours (B) detected in the indicated genotypes at the time of euthanasia (25 weeks after the adenoviral infection). Each dot represents one mouse ( n = 12 animals per genotype analysed). (C) Representative images of tumour‐harbouring lungs from the indicated genotypes at the time of euthanasia (25 weeks after the adenoviral infection) after staining with antibodies to CC10 (upper panels) and SPC (bottom panels). n = 3 independent tissue sections per genotype, 5 animals. Scale bars, 500 (left panels) and 100 (right panels) μm. (D, E) Example (D) and quantification (E) of the proliferation in tumours derived from the indicated genotypes at the time of euthanasia (25 weeks after the adenoviral infection) ( n = 3 independent tissue sections per genotype, 5 animals). Sections were decorated with phospho‐H3 (pH3). Scale bars, 100 μm. (F) Representative images of tumour‐harbouring lungs from the indicated genotypes at the time of euthanasia (25 weeks after the adenoviral infection) after staining with an antibody to phospho‐ERK1/2 ( n = 3 sections per tumour, 5 animals). Scale bars, 500 (left panels) and 100 (right panels) μm. (G, H) Example (G) and quantification (H) of the ICN1 immunostaining (upper panels, green colour) of lung tumours derived from the indicated genotypes at the time of euthanasia (25 weeks after the adenoviral infection) ( n = 2 sections per tumour, 4 animals). In all cases, the nuclei of cells were labelled with DAPI (bottom panels, blue colour). Scale bars, 500 (left panels) and 100 (right panels) μm. Data information: In panels A, B, E, and H, values are shown as mean ± SEM from three independent experiments. Statistical values obtained using the Mann–Whitney U test are given relative to control mice (LSL‐ Kras G12D/+ ). * P ≤ 0.05; ** P ≤ 0.01, *** P ≤ 0.001. [Colour figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: For intracellular ICN1 staining, cells were fixed with Cytofix/Cytoperm (Cat. No. 554714; BD Bioscience, Franklin Lakes, NJ, USA) for 10 min and stained with PE‐labelled antibodies to either TOX (Cat. No. 12‐6502‐82; eBiosciences; 1 : 50 dilution) or ICN1 (mN1A, Cat. No. 552768; Cell Signaling Technologies; 1 : 50 dilution) for 1 h at room temperature in phosphate‐buffered saline solution supplemented with 5% fetal bovine serum and 10% saponin.

Techniques: Infection, Staining, Derivative Assay, Immunostaining, MANN-WHITNEY