Additional file 1 of Proteomic interrogation of the meninges reveals the molecular identities of structural components and regional distinctions along the CNS axis
Creators: name: Santorella, Elise
name: Balsbaugh, Jeremy L.
name: Ge, Shujun
name: Saboori, Parisa
name: Baker, David
name: Pachter, Joel S.
Publisher: name: figshare
Year: 2024
Subjects: subject: Biophysics
subject: Biochemistry
subject: Medicine
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Neuroscience
subject: Physiology
subject: Pharmacology
subject: Biotechnology
subject: Biological Sciences not elsewhere classified
subject: Developmental Biology

description: Additional file 1: Fig. S1. Removal of meninges from mouse brain and spinal cord. Following initial perfusion with PBS to remove blood, Evans Blue dye was perfused to stain the meningeal vessels. (Top row) Brain, with meninges containing stained blood vessels. (a) Before removal of brain meninges. (b) After removal of meninges from the left side of the brain; the right side of the brain shows meninges still adherent. (Bottom row) Spinal cord. (c) Before removal of meninges. (d) After removal of meninges from the caudal end of the spinal cord (left side of image); spinal meninges are shown still adherent to the rostral end. Fig. S2. Compartmental analysis of shared and unique proteins in brain and spinal cord (SC) meninges. Total Proteins from proteomes in Figure 2 were further subdivided into the respective subcellular compartments and numbers of shared and unique proteins within each compartment of brain and spinal cord meninges indicated. Fig. S3. Meningeal trabeculae form a 3D meshwork. Iso-surface rendering (Imaris software) of collagen II-stained trabeculae, highlighting a dense meshwork that permeates the subarachnoid space. This could act to filter leukocytes in the CSF, and aid in nucleating formation of mELTs. As lymphoid chemokines can bind collagen peptides, trabeculae may help set-up concentration gradients necessary to drive immune cell recruitment. Fig. S4. Vimentin and collagen II show divergent networks in the SAS. Collagen II staining within normal mouse spinal meninges casts a diffuse net through the SAS, and is distinct from the focal vimentin staining highlighting trabecular cells. Select cells of the arachnoid and pial layers exhibit staining of both structural proteins. Fig. S5. Immunofluorescence of brain meninges. Sections were cut through whole mouse skull, at a region near the junction of the cerebrum and cerebellum, and adhered to adhesive tape. Brain meninges remain intact as in sections through spinal column (Figs. 4 and 5). (a) Staining of collagen II and collagen III within an apparent dural fold. (b) Staining of ECM protein, tenascin-R, and cytoskeletal protein, vimentin. Fig. S6. IMC of meninges. A section of normal mouse spinal meninges adhered to adhesive tape and processed by IMC as described in Methods. Metal-conjugated antibodies (collagen I and MHC II) and a cationic nucleic acid intercalator containing natural abundance iridium 191Ir and 193Ir) were used. Collagen staining is most prominent in vertebral bone but is also seen along the pia. MHC II identifies some antigen presenting cells in the vertebral bone marrow, while the DNA intercalator highlights a high density of nuclei in the bone. Fig.S7. SEM of subarachnoid trabeculae. Sections of normal mouse spinal meninges adhered to adhesive tape and processed for SEM as described in Methods, highlighting different shaped trabeculae with attached structures. (a) Filiform (←), rod-like (✻), and tree-like (◂) trabeculae. (b) Sheet-like structures (←) between the bone (B), dura (DM) and pia (PM), the latter having been torn away from the underlying parenchyma. (c) Veil-like trabeculae (✻) and what may be trabeculae-associated cell bodies (←). Fig. S8. Immuno-SEM of collagen II. Sections of normal mouse spinal meninges adhered to adhesive tape and processed for immuno-SEM as described in Methods. Fig. S9. Western blotting of meningeal proteins. Western blotting was carried on a sampling of meningeal proteins to compare with proteomic and immunohistological results. Gel images highlighting the respective protein bands are depicted on the left, and corresponding quantification of protein bands is shown on the right. Boxes are denoted for ease of grouping like samples together. For each protein being assessed, three samples each of brain and spinal cord meninges, respectively, were run on the same gel and blotted onto the same membrane. (a) Type III collagen; (b) Type VI collagen; (c) Keratin 8; (d) Type I collagen. Table S1. Primary antibodies used for immunofluorescence, immune-SEM, and Western. Table S2. Secondary antibodies used for immunofluorescence.