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Additional Supporting Information may be found in the online version of this article.

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sc-12-0430_sm_SupplFigure1.pdf2820KFigure S1. Flow cytometric characterization and differentiation potential of MSCs harvested from compact bone. Cells were obtained from tibias and femurs of HO-1+/+ (A) and HO-1−/− (B) mice. Representative light microscope images of MSCs (C) grown in various media to promote differentiation towards osteoblasts (left panel, Alizarin Red S stain), adipocytes (middle panel, Oil-red-O stain, 20x objective) and chondrocytes (right panel, toluidine blue stain, 20x objective).
sc-12-0430_sm_SupplFigure2.pdf1213KFigure S2. MSCs harvested from compact bone. A) Phase contrast images of HO-1+/+ and HO-1−/− MSCs, and B) colony formation (CFU-F) of MSCs after isolation from compact bone. Cells were grown for 14 days in culture and stained with 1% crystal violet. C) Representative Western blot depicting absence of HO-1 protein from MSCs obtained from HO-1−/− mice. D) Flow cytometric characterization of mesenchymal markers in HO-1+/+ (black bars) and HO-1−/− (gray bars) MSCs, using mean fluorescence intensity. .P<0.05 versus HO-1+/+ MSCs, using Student's unpaired t-test. E) Quantitation of osteoblast differentiation in HO-1+/+ (black bar) and HO-1−/− (gray bar) MSCs compared with fibroblasts (Fb, white bar). For the MSCs, the differentiation assays were performed on a minimum of 3 replicates, from at least 3 separate cell isolations. .P<0.05 versus fibroblasts, using one-way analysis of variance followed by Newman-Keuls post test analysis. NS, not significant.
sc-12-0430_sm_SupplFigure3.tif1885KFigure S3. Adhesion molecules are induced in MSCs by pro-inflammatory stimuli (PIS). MSCs obtained from HO-1+/+ (A) and HO-1−/− (B) mice were stimulated with mIL-1β (10 ng/mL), mIFN-γ (10 ng/mL) and mTNF-α (10 ng/mL) for 24 hours. Flow cytometric analysis of the cells was then performed for CD54 and CD106. Bar graphs are depicted as mean±SEM of 3 independent experiments. *P<0.05 versus no (−) PIS, using Student's unpaired t-test. Representative histograms are above each bar graph, with isotype antibody depicted in blue, vehicle (PBS) in red, and PIS in black.
sc-12-0430_sm_SupplFigure4.tif1529KFigure S4. Lung fibroblasts. Flow cytometric characterization of fibroblasts obtained from the lungs of wild-type mice, and depleted of hematopoietic lineage and Sca1. Representative histograms are shown using antibodies for CD140b, CD105, CD44, CD29, CD90, CD73, CD45 and Sca1 in blue, and their isotype antibodies in black.
sc-12-0430_sm_SupplFigure5.tif2451KFigure S5. Assessment of circulating neutrophils, and the effect of Ly6G antibody on neutrophil depletion in wild-type mice. A) Representative flow cytometric pseudo-color density plots and histograms of peripheral blood harvested from control mice demonstrating the percentage of neutrophils (Gr-1+) and macrophages (F4/80+) that are found within the gated area (purple oval) of SSC versus FSC pseudo-color density plots. B) Treatment with the Ly6G (clone1A8) antibody results in depletion of circulating neutrophils indicated by a decreased number of cells in the gated area of SSC versus FSC pseudo-color density plots, detected at 12 hours post injection and persisting through 72 hours. The bar graphs represent the effects of Ly6G antibody (white bars), or IgG control (black bars), on the percentage of circulating neutrophils after 12 and 72 hours (3 independent experiments). Data is presented as mean±SEM. *P<0.05 versus IgG control, using Student's unpaired t-test.
sc-12-0430_sm_SupplFigure6.tif2819KFigure S6. MSCs enhance neutrophil phagocytosis, and do not phagocytize bacteria alone. A) Peritoneal neutrophils activated with mG-CSF were incubated with GFPlabeled E. coli or E. faecalis in the presence of MSCs (HO-1+/+ or HO-1−/−) or in the absence of MSCs (− MSCs). Representative flow cytometric pseudo-color density plots assessing phagocytosis of GFP-labeled E. coli and E. faecalis bacteria by neutrophils. B) MSCs from HO-1+/+ and HO-1−/− mice activated with a pro-inflammatory stimulus (mIL-1β, mIFN-γ, and mTNF-α) and incubated with GFP-labeled E. coli or E. faecalis bacteria at 10 MOI (10 bacteria per 1 MSC). After two hours, cells were harvested and stained using a CD140b antibody to detect MSCs, and assessed for bacterial phagocytosis.
sc-12-0430_sm_SupplFigure7.tif422KFigure S7. Circulating WBCs and organ Gr-1+ cells are decreased by MSCs during CLP-induced polymicrobial sepsis. A) White blood cell counts in HO-1-/- mice after sham (−) or CLP (+) surgery, following treatment with MSCs or fibroblasts, n=9-11 per group. Horizontal bars represent mean values. *P<0.05 versus sham; †P<0.05 versus fibroblasts, using one-way analysis of variance followed by the Newman-Keuls post test analysis. Quantitative analysis of Gr-1+ cells in the small bowels (B), kidneys (C,) and livers (D) of HO-1−/− mice 24 hours after sham surgery or CLP. Data are expressed as mean±SEM, n=9-10 per group. *P<0.05 versus sham; †P<0.05 versus fibroblasts, using Kruskal-Wallis one-way analysis of variance followed by the Dun post test analysis.
sc-12-0430_sm_SupplMethods.pdf19KSupplementary Data
sc-12-0430_sm_SupplTable1.tif1485KSupplementary Table 1

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