Elastic fiber, collagen and pSMAD2 staining
Collected aortic tissue was embedded in paraffin and cut into 5µm thick sections. Elastic fiber integrity and collagen content were assessed using Verhoeff Van Gieson (Sigma-Aldrich) and Trichrome Masson’s (Sigma-Aldrich), respectively, according to manufacturer’s instructions. pSMAD2 was visualized using immunohistochemistry. Deparaffinization in toluene (5 minutes) was followed by rehydration in 100%, 90%, 70% and 50% ethanol solutions, after which sections were subjected to 3% hydrogen peroxide followed by incubation in trypsin for 10 minutes at 37 °C (Sigma-Aldrich, 93615-25G). Sections were heated in citrate buffer for 10 minutes at 90°C and blocked for 20 minutes at room temperature (RT) with goat serum (Vector-lab consult). A primary pSMAD2 antibody (3101, Cell Signaling; 1:5000) was incubated overnight using a concentration of 1:5000 and the following day, the sections were incubated with the secondary antibody (30014 secondary goat anti-rabbit IgG, Vector; 1:200) for 30 min and subsequently with an avidin-biotinylated complex for 1 hour, both at RT (Vectastain ABC kit, Vector Laboratories). 3,3-Diaminobenzidine tetrahydrochloride hydrate (DAB) chromogen (Sigma-Aldrich) was used as the substrate and Hematoxylin as counterstaining. All pictures were acquired using a Leica DMi8 inverted microscope, at a 20x magnification.
Figure 1: Pedigrees of two families with JAG1 variants. A) family with p.Cys748Arg variant; B) family with the c.2114-5_2119delins18 variant.
Figure 2: Histological staining of elastin (A and B), collagen (C and D), and phosphorylated SMAD2 protein (pSMAD2, brown nuclear staining, E and F) of aortic tissue of a healthy control (top) and patient A:III:11 (bottom). Scalebar: 100 µm.
Figure S1: Splicing analysis of the insertion-deletion variant encompassing the splice acceptor site and first nucleotides of exon 17 (NM_000214.2: c.2114-5_2119delins18).