Fig 2: Notch1 assembles a mechanosensory junctional complex involving LAR, Trio, and Raca, Fluorescent micrographs of phalloidin stained ECs. b, Intensity of cortical actin at cell-cell junctions and the number of stress fibers per micron quantified from phalloidin stained ECs (n=3 hEMVs). c, Active Rac1 was isolated from scramble and Notch1-KO cell lysates using a recombinant p21-binding domain of Pak1 (GST-PBD). d, Quantification of western blot band intensity revealed a decrease in Rac1 (~50%) after knockout of Notch1 (n=3 independent lysates). e, Active Rac1 was isolated using GST-PBD from Notch1-KO cell lysates expressing mApple or TMD-mApple. f, Quantification of western blot band intensity revealed an increase in Rac1 (~45%) with expression of TMD-mApple (n=3 independent lysates). g, PD of Notch1-KO ECs expressing TMD-mApple or mApple control in the presence of 50 µM NSC 23766, a Rac1 inhibitor, or vehicle control. h, Immunoprecipitation of VE-cadherin and the Rac1 GEF Trio from ECs cultured statically or under flow. Co-immunoprecipitation of mechanosensory complex proteins was assessed by immunoblotting for Notch1-ECD, Notch1-ICD, LAR (85 kDa P-subunit), and VE-cadherin. i, Immunoprecipitation of VE-cadherin or Trio from scramble and Notch1-KO ECS cultured under flow. Co-immunoprecipitation of mechanosensory complex constituents was assessed by immunoblotting for VE-cadherin, LAR, and Trio. j, PD of LAR-KO and Trio-KO ECs cultured under static or flow conditions or in the presence of rDll4-coated collagen. k, Micrographs of Trio-KO and LAR-KO cells under flow conditions (VE-cadherin – magenta, actin – green, DAPI – blue). l, Quantification of junctional area measured from VE-cadherin immunostained micrographs. m, Immunoprecipitation of VE-cadherin and Trio from Notch1-KO cells expressing TMD-mApple or mApple. Immunoblotting with LAR, VE-cadherin, and RFP antibodies was used to assess co-immunoprecipitation of mechanosensory complex upon expression of TMD. n. PD for scramble, N1KO, LAR-KO, and Trio-KO cells cultured under static conditions expressing TMD-mApple or mApple infection control. For (g,j,n), n≥3 hEMVs. All plots mean ± s.e.m., *p<0.05, **p<0.01. Exact p and n values available in Figure Source Data, all images representative of at least three independent experiments.

Fig 3: Notch1 regulates junctional stability through association with VE-cadherina, Timelapse images of cells expressing VE-cadherin-mApple in the presence of DAPT or DMSO load control demonstrate that AJs disassemble after 30min of exposure to DAPT, leading to macroscopic intercellular gaps (red arrows). b, Fluorescent micrographs of Notch1 KO cells expressing TMD+ICD-mApple or TMD+ICD V1754G-mApple immunostained for cleaved Notch1 ICD V1754 and DAPI. c, Fluorescent micrographs of TMD-mApple expressed in VE-cadherin knockout or scramble control ECs and immunostained for VE-cadherin. d, Western blot for Notch1 ICD and VE-cadherin in Notch1-KO and VE-cadherin KO ECs. e, Western blot validation of Notch1 rescue constructs: mApple, TMD-mApple, ICD+TMD-mApple, and ICD+TMD V1754G-mApple. f, Immunoprecipitation of VE-cadherin from hMVEC-D cells treated with DMSO or DAPT. Co-immunoprecipitation of mechanosensory complex components was assessed by immunoblotting for Notch1 ICD, Trio, and LAR. g. Western blot of VE-cadherin immunoprecipitations from Notch1 KO cells expressing Notch1-TMD truncation constructs (6, 8, 12 amino acids from the N-terminus) fused to mApple. h, Western blot of VE-cadherin immunoprecipitations from Notch1 KO cells expressing single and dual point mutation Notch1-TMD constructs (within the transmembrane segment of Notch1 TMD) fused to mApple. All images representative of at least three independent experiments.

Fig 4: Notch1 regulates VE-cadherin interacting proteins to form the Notch1 mechanosensory complexa, Immunoprecipitation of VE-cadherin from scramble and Notch1-KO cells. Co-immunoprecipitation of candidate Notch1-dependent, VE-cadherin effectors was assessed by immunoblotting for VE-PTP, VEGFR2, and LAR (85 kDa P-subunit). b, Immunoprecipitation of the Rac1 GEF Trio from scramble, Notch1-KO, and LAR-KO cells. Immunoblotting for VE-cadherin was used to assess impaired Trio-VE-cadherin co-immunoprecipitation upon depletion of Notch1 or LAR. c, Western blots of VE-cadherin immunoprecipitated from the lysates of lungs from CDH5-Cre(+);NOTCH1fl/fl and control CDH5-Cre(+);NOTCH1fl/fl mice and immunoblotted for LAR. d, Western blots of Trio immunoprecipitated from the lysates of lungs from CDH5-Cre(+);NOTCH1fl/fl and control CDH5-Cre(+);NOTCH1fl/fl mice and immunoblotted for VE-cadherin. e, Western blot of proximal interacting proteins extracted with streptavidin from hMVEC-D cells expressing BirA-HA (BioID) or VE-cadherin-BirA-HA (VE-BioID) that were treated with DMSO, Dll4, or DAPT, immunoblotted for HA and Notch1 ICD. f, Western blot of proximal interacting proteins extracted with streptavidin in hMVEC-D cells expressing VE-cadherin-BirA-HA (VE-BioID) that were treated with DMSO, Dll4, or DAPT, immunoblotted for Trio and LAR. All images representative of at least two independent experiments.

Fig 5: Dll4 and the Notch1 mechanosensory complex are critical for increased Rac1 activity in response to shear stressa, Active Rac1 was isolated with GST-PBD from hMVEC-D cell lysates treated with DMSO and DAPT (20 µM). b, Quantification of western blot band intensity demonstrates a decrease (~30%) in Rac1 activity with DAPT treatment. c, Active Rac1 was isolated using GST-PBD from hMVEC-D cell lysates from static or shear flow conditions. d, Active Rac1 was isolated using GST-PBD from Dll4-KO cell lysates under flow conditions. e, Active Rac1 was isolated using GST-PBD from Notch1 KO, LAR KO, and Trio KO cell lysates under flow conditions. Mean ± s.e.m., n=3 independent lysates, **p<0.01. Exact p and n values available in Figure Source Data, all images representative of at least three independent experiments.