Additional file 1 of CD146 expression profile in human skin and pre-vascularized dermo-epidermal skin substitutes in vivo
Creators: name: Nanni, Monica
name: Rütsche, Dominic
name: Bächler, Curdin
name: Pontiggia, Luca
name: Klar, Agnes S.
name: Moehrlen, Ueli
name: Biedermann, Thomas
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Space Science
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer
subject: Hematology
subject: Infectious Diseases
subject: FOS: Health sciences
subject: Virology
subject: Computational Biology

description: Additional file 1: Supplementary Fig. S1. PLVAP expression on human blood capillaries. A, B, C, and D Representative immunofluorescence images of human skin sections co-stained for PLVAP with CD31, or with the markers of lymphatic endothelial cells, Lyve1, Podoplanin, Prox1. PLVAP co-localize with CD31 (A), but it is not expressed on lymphatic vessels (B, C, D). Scale bar:50 μm. E Individual channels of the immunofluorescence stainings shown in Figure 1E for human skin sections stained for CD146, PLVAP, CD31. (n = 3 independent donors). Scale bar: 50 μm. Supplementary Fig. S2. Individual channels of immunofluorescence stainings in Figure 2. A, B, and C Individual channels of the immunofluorescence stainings shown in Figure 2 for human skin sections stained for CD146, CD31 and Podoplanin (A) or Lyve1 (B) or Prox1 (C). (n = 3 independent donors). Scale bar: 50 μm. Supplementary Fig. S3. Individual channels of immunofluorescence stainings in Figure 3. A, B, and C Individual channels of the immunofluorescence stainings shown in Figure 3 for human skin sections stained for CD146, CD31 and NG2 (A) or desmin (B) or αSMA (C). The dotted line represents the dermo-epidermal junction. (n = 3 independent donors). Scale bar: 50 μm. Supplementary Fig. S4. Gating strategy for flow cytometric analysis of freshly isolated HDMECs from human foreskin dermis. A The gates have been established based on isotype controls, unstained and single stained cells. Fluorescence-minus-one (FMO) controls were performed to defined the positive gates. B Hierarchical gating strategy performed to separates BECs and LECs. Gates were settled to consecutive exclude debris, doublets, and dead cells. Staining with CD31-PE and Podoplanin-A488 allowed to sort HDMECs into BECS (CD31+Podoplanin-) and LECs (CD31+Podoplanin+). The two cell populations have been reanalyzed by flow cytometry to confirm their purity. (n = 3 independent donors). Supplementary Fig. S5. Gating strategy for flow cytometric analysis of freshly isolated cells from human dermis. The gates have been established based on unstained and single stained cells. Fluorescence-minusone (FMO) controls were performed to define the positive gates. (n = 3 independent donors). Supplemental Fig. S6. Cell viability of pericytes and BECs after stimulation with cytokines. A, B CD146+ pericytes and BECs were left untreated or stimulated with TNFα, IL-1β and IL-6 for 12h, 24h and 48h. Live/dead staining with fluorescein diacetate–propidium iodide (FdA–PI, FdA in green, PI in red) was performed at the indicated time points to assess cell viability. Scale bar: 100 μm. (n=3). Supplementary Fig. S7. CD146 expression does not appear in LECs stimulated with proinflammatory cytokines. LECs were left untreated or stimulated with TNFα, IL-1β, and IL-6 for 12h, 24h, and 48h. Western blot analysis shows no detection of CD146 expression in both control condition and after stimulation with TNFα, IL-1β, and IL-6. Unstimulated BECs were used as positive control for CD146 expression. The equal loading was assessed using anti-GAPDH. (n = 3 independent donors). Supplementary Fig. S8. CD146/CD90 expression on cells in a 3D-prevascularized hydrogel. Cells in 3D collagen type I hydrogel are stained for CD146, CD90 and CD31. Representative confocal images show CD146/CD90-double positive pericyte-like cells surrounding blood capillaries. Single CD90-positive cells identify fibroblasts. (n = 3 independent donors). Scale bar: 100 μm, inset 50 μm. Supplemental Fig. S9. CD146 expression in prevascularized dermo-epidermal skin substitutes in vivo. A, B CD146 expression is detected on CD31-positive capillaries and on pericytes-like cells investing these capillaries (A), but CD146 is not present on Prox1-positive lymphatic capillaries (B). White dashed lines indicate the dermo-epidermal junction. Scale bar 100 μm, inset 50 μm. C CD146 colocalizes with NG2 in human pericytes associated to CD31-positive capillaries. Scale bar: 100 μm, inset 50 μm. D The co-localization between CD146 and humanCD31 and the absence of CD146 expression on rat capillaries confirm the specificity of the CD146 antibody for human capillaries. (n = 3 independent donors). Scale bar: 100 μm, inset 50 μm. Dotted line represents dermo-epidermal junction. Supplementary Table 1. List of antibodies The following primary and secondary antibodies were used for immunofluorescence (IF), whole mount (WM), western blot (WB), FACS (FC).