Journal: bioRxiv
Article Title: Targeting NEDD9-SH3 with a Covalent Peptide Controls Endothelial Phenotype
doi: 10.1101/2025.07.10.663547
Figure Lengend Snippet: a. Paraffin-embedded lung sections from control, rat SU-5416-Hypoxia-Normoxia-PAH, and rat monocrotaline (MCT)-PAH ( n =4-5 rats/condition) or isolated from idiopathic PAH patients ( n =5-8/condition) at the time of lung explant were stained using anti-PECAM1 and anti-NRP-1 antibodies and the immunohistochemical profile in distal pulmonary arterioles was analyzed. Scale bar, 20 μm. b. Human pulmonary artery endothelial cells (HPAECs) and human pulmonary artery smooth muscle cells were co-cultured in a barrier-free system to recapitulate the direct cell-cell contact conditions in PAH lesions. Double immunogold-labeled staining was performed using anti-NRP-1 and anti-Heparan sulfate antibodies, and imaged using transmission electron microscopy. Scale bar, 500 nm. Inset scale bar, 100 nm. c. Disease prevention protocol using the rat SU-5416-hypoxia-normoxia and monocrotaline (MCT) experimental models of PAH and timepoints for intratracheal administration of si-Scrambled (negative) control (Scr) or siRNA against NEDD9. d. The right ventricular systolic pressure (RVSP) measured by cardiac catheterization ( n =3-7 rats/condition) and e. RV mass measured by Fulton Index. IVs, interventricular septum; LV, left ventricle. f. Human pulmonary artery endothelial cells were untreated or treated with VEGF-C (100 nM) for 6 hr and transfected with vehicle (V) control or adenovirus (Ad) containing green fluorescent protein (GFP), Sulf1 cDNA, or siRNA-Sulf1, and cell migration was quantitated using the wound healing assay. Black line, separate experiment iteration without VEGF-C or other treatment ( n =4-7). Blue, DAPI. Scale bar, 400 μm. g. HPAECs were untransfected or transfected with an Ad carrying vehicle (V) control, Scr, Sulf1, or si-Sulf1 and stained with an anti-FAK-Y861 antibody. The number of podosome rosette-positive cells (podosome+) per low power field (40x) and the number of cells with >2 podosomes was quantitated ( n =3). Arrows indicate podosome structures. Scale bar, 50 μm. h. The number of tubes formed per 10x field was counted for untreated HPAECs and cells transfected with Ad carrying V, Sulf1, and si-Sulf1 ( n =3). Scale bar, 300 μm. i . The effect of Ad-Sulf1 on collagen III deposition in HPAECs ( n =5-10). PFE, plaque forming unit (N=1-3 PFEs). Scale bar, 50 μm.
Article Snippet: HPAECs were grown on glass chamber slides (Lab-Tek II) and transfected for 24 hours with adenovirus expressing GFP (Ad-GFP), GFP plus human Sulfatase-1 (Ad-Sulf1) or GFP plus human Sulf1 siRNA (Ad-siSulf1), 1 PFU/ml x10 7 (Vector Biolabs) and stained with TRITC-conjugated phalloidin for actin and following antibodies: phospho-FAK (Tyr861), phospho-FAK (Tyr576), vinculin, cortactin, sulfatase-1 and neuropilin-1.
Techniques: Control, Isolation, Staining, Immunohistochemical staining, Cell Culture, Labeling, Transmission Assay, Electron Microscopy, Negative Control, Transfection, Migration, Wound Healing Assay
Vector Biolabs is a verified supplier 
Vector Biolabs manufactures this product 
