Additional file 1 of Preadipocytes in human granulation tissue: role in wound healing and response to macrophage polarization
Creators: name: Rauchenwald, Tina
name: Handle, Florian
name: Connolly, Catherine E.
name: Degen, Antonia
name: Seifarth, Christof
name: Hermann, Martin
name: Tripp, Christoph H.
name: Wilflingseder, Doris
name: Lobenwein, Susanne
name: Savic, Dragana
name: Pölzl, Leo
name: Morandi, Evi M.
name: Wolfram, Dolores
name: Skvortsova, Ira-Ida
name: Stoitzner, Patrizia
name: Haybaeck, Johannes
name: Konschake, Marko
name: Pierer, Gerhard
name: Ploner, Christian
Publisher: name: figshare
Year: 2024
Subjects: subject: Biophysics
subject: Biochemistry
subject: Microbiology
subject: FOS: Biological sciences
subject: Cell Biology
subject: Molecular Biology
subject: Physiology
subject: Pharmacology
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Cancer

description: Additional file 1: Supplement Figure 1. CD34expression within cell clusters in human wound tissue. Expression of CD34 in fibroblast cell clusters and proportion of cells originating from diabetic foot ulcer (DFU) healers compared to non-healers for each cluster. Left panel: CD34 expression in CD34- and CD34+ fibroblast cell clusters split by wound healing response. Right panel: Expression of CD34 in all identified fibroblast subpopulations showing high CD34 expression in three clusters (Fb_2, Fb_6, Fb_7). Data are shown as violin plots. Supplement Figure 2. CD34+ and CD31+ cells repopulate human granulation tissue. Immunohistochemistry of a representative section of granulation tissue from a chronic lower limb wound in a 53-year-old male patient stained for CD34 (pink) and CD31 (brown). Left image: Cross-sectional view of the granulation tissue specimen. Scale bar indicates 1 mm. Right image: Detailed view of the same specimen showing the distribution of CD34+ cells (black arrows). Scale bars indicate 100 µm. Supplement Figure 3. Flow cytometry gating strategy. Flow cytometry gating strategy that discriminates single cell populations to define CD45-/CD31-/CD90+/CD34+ adipocyte precursor cells. Supplement Figure 4. Flow cytometric analysis. Flow cytometric analysis of single cell suspensions from human granulation tissue samples (n = 6). Mean frequencies of cell types are shown as a percentage of total viable granulation tissue cells. Supplement Figure 5. Effect of MQ-CM on ASC size and viability. Data from ASCs cultured in differentially activated MQ-CM for 72 h are shown as fold change compared to monocyte-CM treatment (Mo) (n = 5). Cell volume and diameter were analyzed using CASY TT cell counter. Cell viability corresponds to the number of cells negative for Annexin V and 7AAD staining as assessed by flow cytometry. Data are shown as mean ± SEM. Asterisks indicate p-values of &lt;0.01 (**). Supplement Figure 6. Comparison of IL1B- and TGFB1-mediated effects on ASC physiology. (A) Representative confocal microscopy images of ASCs cultured in the presence of recombinant human IL1B (10 ng/ml) or TGFB1 (10 ng/ml) for 72 h and analyzed for actin cytoskeleton remodeling. Scale bars indicate 20 µm. (B) Quantification of mitochondrial membrane potential (MMP) (n = 4). Data are shown as mean ± SEM. Asterisks indicate p-values of p &lt; 0.01 (**). (C-E) Representative immunoblots of cell lysates of the respective cells analyzed for cell adhesion proteins (C), intracellular kinases (D), or glycolysis regulating enzymes (E). Supplement Figure 7. Efficacy of kinase inhibition. (A) Representative immunoblots of ASCs exposed to different MQ-CM for 1 h. Kinase inhibition of M1-CM treatment with kinase inhibitors Ly294001 (PI3K/AKT), U0126 (ERK1/2), and Losmapimod (p38-MAPK) demonstrated efficacy. (B) Representative microscopic images of phalloidin staining (green) for actin stress fibers in ASCs cultured in M1-CM supplemented with LY294001 (10 µM), U0126 (10 µM) or Losmapimod (10 µM) for 72 h.