Additional file 1 of Serpin-loaded extracellular vesicles promote tissue repair in a mouse model of impaired wound healing
Creators: name: Park, Dong Jun
name: Duggan, Erika
name: Ho, Kayla
name: Dorschner, Robert A.
name: Dobke, Marek
name: Nolan, John P.
name: Eliceiri, Brian P.
Publisher: name: figshare
Year: 2022
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Molecular Biology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: Immunology
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Cancer
subject: Computational Biology

description: Additional file 1: Figure S1. Statistical analysis of CD marker expression in PVA infiltrates at various time points. In support of the piechart analyses in Fig. 1, immune cells infiltrating PVA sponges were quantified at each time point focusing on: a Macrophages, b Inflammatory monocytes, c Neutrophils, d Dendritic cells, and e T cells (p-value: ***&lt;0.001, **&lt;0.005, *&lt;0.05). Figure S2. Evaluation of EV biogenesis related gene expression in the PVA cells by qRT-PCR depending on the 2, 7, and 14 days. Measurements of changes in gene expression normalized to GAPDH in cells infiltrating PVA sponges focusing on the following canonical biogeneis genes: a Rab5a, b Rab5b, c Rab27, d Rab27b e Rab11a, f VPS4a, g TSG101, and h Rab35 (p-value: **&lt;0.005, *&lt;0.05). Figure S3. Analysis of baseline cell infiltrate in PVA sponges of WT vs. db/db mice, from which EVs were harvested. Flow cytometry analysis of immune cells recruited to the PVA sponge at 14 days post-implantation focusing on : a monoyctes based on CD11b+Ly6Chigh cells as a subset of CD45+ cells, b neutrophils based on Ly6G+Ly6Clow cells as a subset of CD45+ cells, c macrophages based on CD11b+F4/80+ cells as a subset of CD45+ cells, d DCs based on MHCII+ cells as a subest of CD11b+CD11c+ cells, e T cells based on CD4+ cells as a subset of CD3+ cells, f CD8+ T cells as a subset of CD3+ cells, and g Regulatory T cells based on CD4+CD25+ as a subset of CD3+ cells. (p-value: *&lt;0.05). Figure S4. Validation of EVs derived from WT and db/db mice by vFRed analysis. a Size distribution of WT EVs. b Plot diameter data of WT EVs. c Expression of Annexin V-PE on WT EVs. d Expression of tetraspanins CD9, CD63 and CD81 (TS-PE)on WT EVs. e Histogram of Annexin V-PE expression on WT EVs normalized to buffer only (grey filled in). f Histogram of TS-PE on WT EVs normalized to buffer only (grey filled in). g Size distribution of db/db EVs. h Plot diameter data of db/db EVs. i Expression of Annexin V-PE on db/db EVs. j Expression of TS-PE on db/db EVs. k Histogram of Annexin V-PE expression on db/db EVs normalized to buffer only (grey filled in). l Histogram of TS-PE on db/db EVs normalized to buffer only (grey filled in). Figure S5. Validation of lentivirus titer testing using HEK293 cells. a Representative comparison of lentivirus yields b based on titer testing of lentiviral p24 protein using GoStix analysis per manufacturers recommendations. Figure S6. A representative example of the analysis of EVs engineered to express specific proteins. a EVs collected from the conditioned media of parental cells as a negative control (Left, naïve HEK293 donor cells) and enriched by density ultracentrifugation were subjected to vFRED staining to determine EV diameter as described in the Materials and Methods. GFP-loaded EVs (XP-GFP) in absence (Left) or presence (Right) of anti-CD81-PE tetraspanin were analyzed for b GFP fluorescence and c expression of CD81. d and e An overview and quantification of GFP-EV internalization into HEK293 cells (filled in) compared to buffer control (open). Figure S7. Quantification of K14 immunohistochemistry. The fluorescent intensity of ant-K14 stained tissue sections from Figure 5l were quantified using Image J. A minimum of 3 fields were analyzed for each of the EV treated samples (p-value: * &lt;0.05). Table S1. Detail information of primer for extracellular vesicle biogenesis related genes. Table S2. Primer sequences for cloning of XP tag in-frame with SERPINA1, SERPINF2, and SERPING1.