DOI: 10.5061/dryad.m63xsj49c
title: The bone marrow endothelial progenitor cell response to septic infection: Dataset

description: An early increase in the level of endothelial progenitor cells (EPCs) in
 the systemic circulation occurs in patients with septic infection/sepsis.
 The significance and underlying mechanisms of this response remain
 unclear. This study investigated the bone marrow EPC response in adult
 mice with septic infection induced by intravenous injection (i.v.) of
 Escherichia coli. For in vitro experiments, sorted marrow stem/progenitor
 cells (SPCs) including lineage(lin)-stem cell growth factor
 receptor(c-kit)+stem cell antigen-1(Sca-1)-, lin-c-kit+, and lin- cells
 were cultured with or without lipopolysaccharides (LPS) and recombinant
 murine vascular endothelial growth factor (VEGF) in the absence and
 presence of anti-Sca-1 crosslinking antibodies. In a separate set of
 experiments, marrow lin-c-kit+ cells from green fluorescence protein
 (GFP)+ mice, i.v. challenged with heat-inactivated E. coli or saline for
 24 h were subcutaneously implanted in Matrigel plugs for 5 weeks. Marrow
 lin-c-kit+ cells from Sca-1 knockout (KO) mice challenged with
 heat-inactivated E. coli for 24 h were cultured in the Matrigel medium for
 8 weeks. The marrow pool of EPCs bearing the lin-c-kit+Sca-1+VEGF receptor
 2 (VEGFR2)+ (LKS VEGFR2+) and LKS CD133+VEGFR2+ surface markers expanded
 rapidly following septic infection, which was supported by both
 proliferative activation and phenotypic conversion of marrow
 stem/progenitor cells. An increase in marrow EPCs and their reprogramming
 for enhancing angiogenic activity correlated with cell-marked upregulation
 of Sca-1 expression. Sca-1 coupled with ras-related C3 botulinum toxin
 substrate 2 (Rac2) in signaling the marrow EPC response. Septic infection
 caused a substantial increase in plasma levels of IFN-γ, VEGF, G-CSF, and
 SDF-1. The early increase in circulating EPCs was accompanied by their
 active homing and incorporation into pulmonary microvasculature. These
 results demonstrate that the marrow EPC response is a critical component
 of the host defense system. Sca-1 signaling plays a pivotal role in the
 regulation of EPC response in mice with septic infection.
description: <strong><em>Animals</em></strong> C57BL/6 and BALB/c mice (6-8 weeks old, both sexes) were purchased from Charles River Laboratories (Wilmington, MA) and Taconic Biosciences, Inc. (Germantown, NY). Breeding pairs of C57BL/6-Tg (UBC-GFP)30Scha/j mice were purchased from the Jackson Laboratory (Bar Harbor, ME). C57BL/6-Tg (UBC-GFP)30Scha/j mice and Sca-1<sup>-/-</sup> mice (Sca-1 knockout or Sca-1 KO considered congenic on the C57BL/6 background, originally transferred from Dr. William L. Stanford’s group at University of Toronto, Toronto, Canada) were bred under specific pathogen-free conditions in the Animal Care Facilities of Northeast Ohio Medical University, Michigan State University, and Louisiana State University Health Sciences Center, respectively. These mice were used for experiments at the age of 6-8 weeks old. All animals were housed in specific pathogen-free facilities with a 12-hour light/dark cycle. Approvals from the Institutional Animal Care and Use Committees in adherence with National Institutes of Health guidelines were obtained before the initiation of experiments. Septic infection was induced in mice as described previously with minor modifications (24-26). Briefly, an intravenous injection (i.v. via the jugular or penile vein) of live or heat-inactivated <em>Escherichia coli</em> (<em>E. coli</em> strain E11775 from the American Type Culture Collection, Rockville, MD, ~1 x 10<sup>7</sup> to ~1 x 10<sup>8</sup> CFUs in 100 μl of pyrogen-free saline/mouse). Control mice were injected with an equal volume of saline. In a subset of experiments, septic infection was induced in mice by i.v. ~1 x 10<sup>8</sup> CFUs of <em>E. coli</em> in 50 μl of saline/mouse. 5-bromo-2-deoxyuridine (BrdU, 1 mg in 100 μl of phosphate-buffered saline, BD Bioscience, San Jose, CA) was i.v. administered simultaneously to each mouse. Animals were sacrificed at scheduled time points as indicated in each figure legend. At the time of sacrifice, a heparinized blood sample was obtained by cardiac puncture. White blood cells (WBCs) were quantified under a light microscope with a hemocytometer. Plasma samples were collected after centrifugation of blood samples at 500 g for 10 min. Peripheral blood mononuclear cells (PBMCs) were isolated using lymphocyte density gradient separation media 1.086 (Cedarlane, Burlington, ON, Canada). Both femurs and tibias were collected. Bone marrow cells (BMCs) were flushed out from these bones with a total volume of 2 ml RPMI-1640 medium (Life Technologies, Grand Island, NY) containing 2% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO) through a 23-gauge needle. BMCs were filtered through a 70-micron nylon mesh (Sefar America Inc. Kansas City, MO). Contaminating erythrocytes in BMC samples were lysed with RBC lysis solution (Qiagen Sciences, Germantown, MD). Nucleated BMCs were washed with RPMI-1640 medium containing 2% BSA and then quantified under a light microscope with a hemocytometer. To generate green fluorescence protein (GFP) expressing bone marrow chimeric mice, recipient wildtype C57BL/6 mice received whole body irradiation at 900 rads/mouse and then were i.v. injected (via the jugular vein) with nucleated bone marrow cells (5 x 10<sup>6</sup>/mouse) from donor C57BL/6-Tg (UBC-GFP)30Scha/j mice under isoflurane anesthesia. Antibiotics (neomycin 1.1 g/liter of water and Polymyxin 1 million unit/liter of water) in drinking water were given to recipient mice 1 week before and 1 week after the bone marrow transplantation. Between 6 to 8 weeks post bone marrow transplantation, a blood sample was obtained from the tail vein of each mouse under isoflurane anesthesia for assessing hematologic reconstitution by counting GFP<sup>+</sup> cells under an Olympus IX81 time-lapse deconvolution fluorescent/phase contrast microscope (Olympus America Inc. Melville, NY). In this set of experiments, GFP<sup>+</sup> white blood cells (WBCs) comprised 92±1% of total WBCs in peripheral blood samples at the time of assessment. Septic infection (i.v. 1 x 10<sup>8</sup> CFUs of <em>E. coli</em> in 100 µl saline/mouse) was induced in GFP bone marrow chimeric mice 12 weeks after bone marrow transplantation to collect lung samples at designated time points for morphological assessment of pulmonary recruitment of marrow cells. In another set of experiments, C57BL/6-Tg (UBC-GFP)30Scha/j mice received i.v. challenge with either heat-inactivated (60°C for 60 min) <em>E. coli</em> (1 x 10<sup>8</sup> CFUs in 100 µl saline/mouse) or an equal volume of saline for 24 h. Marrow lin<sup>-</sup>ckit<sup>+</sup> cells are sorted and seeded in ice-cold HC Matrigel (BD Bioscience) containing 50 ng/ml recombinant murine VEGF (BioLegend, San Diego, CA) at 1 x 10<sup>5</sup> cells/ml. Matrigel (0.5 ml of Matrigel containing 50,000 cells/pouch) was implanted subcutaneously into each recipient wildtype C57BL/6 mouse for 5 weeks. Implanted Matrigel plugs were then collected for analyzing vasculogenic activity in donor GFP<sup>+</sup> marrow lin<sup>-</sup>ckit<sup>+</sup> cells. <strong><em>Preparation of bacteria</em></strong>          For each experiment, a frozen stock culture of <em>E. coli</em> was added to tryptic soy broth and incubated for 18 h at 37°C in an orbital shaker. Bacteria were collected and washed twice with phosphate-buffered Saline (PBS, Thermo Scientific, Frederic, MD). Suspension of bacteria in saline at appropriate concentrations was prepared based on its optical density at 600 nm. Actual numbers of viable bacteria were verified by standard plate counts of the bacterial suspensions on MacConkey agar plates following overnight incubation at 37°C. <strong><em>In vitro culture of marrow SPCs</em></strong> Sorted bone marrow lin<sup>-</sup>ckit<sup>+</sup>Sca1<sup>-</sup>, lin<sup>-</sup>ckit<sup>+</sup>, and lin<sup>-</sup> cells from naïve mice were cultured in StemSpan Serum-Free Expansion Medium (StemCell Technologies, Vancouver, BC, Canada) containing 10% of mouse plasma, 100 units-100 µg/ml of penicillin-Streptomycin (Thermo Fisher Scientific, Inc., Rockford, IL) with or without lipopolysaccharides (LPS, <em>E. coli</em> 0111:B4, 20-50 ng/ml, Sigma-Aldrich) and recombinant murine VEGF (50-250 ng/ml, BioLegend) in the absence and presence of anti-Sca-1 crosslinking antibodies (25 µg/ml of each clone D7 and E13, BD Biosciences) for 24 h. In one set of cultures, BrdU (10 µM, BD Biosciences) was added to the culture system during the last 4 h of culture. <strong><em>Determination of late EPC colony-forming activity in marrow SPCs</em></strong> Sca-1 KO and wildtype C57BL/6 mice were i.v. challenged with heated inactivated <em>E. coli</em> (1 x 10<sup>8</sup> CFU in 100 µl saline/mouse) or an equal volume of saline for 24 h. Marrow lin<sup>-</sup>ckit<sup>+</sup> cells were sorted from each animal and cultured in 24-well plates. Each well contained 510 µl of Matrigel culture mixture [3 x 10<sup>4</sup> cells in 510 µl of 2/3 Matrigel-1/3 endothelial progenitor outgrowth cell (EPOC) medium (BioChain Institute Inc., Newark, CA) mixture containing 25 ng recombinant murine VEGF (BioLegend)] overlaid with 200 µl of EPOC medium containing penicillin-Streptomycin (100 units-100 µg/ml). Cell cultures were maintained at 37°C in an atmosphere of 5% CO<sub>2</sub>. The overlaid EPOC medium was changed every 2-3 days during the cultural period of 8 weeks. Late EPC colonies formed in each well were counted under the Olympus IX81 time-lapse deconvolution fluorescent/phase contrast microscope (Olympus America Inc.) at the end of the culture. <strong><em>Flow cytometry and cell sorting</em></strong> Cell phenotype, intracellular expression of specificity protein 1 (SP1) as well as cell BrdU incorporation were determined with flow cytometry as previously described (24-27). Briefly, nucleated BMCs and PBMCs suspended in RPMI-1640 containing 2% BSA (1 x 10<sup>6</sup> cells in 100 μl medium) were added with a mixed panel of biotinylated anti-mouse lineage markers [10 µg/mL of each antibody against CD3e (clone 145-2C11), CD45R/B220 (clone RA3-6B2), CD11b (Mac-1, clone M1/70), TER-119 (clone TER-119)] and granulocyte differentiation antigen 1 (Gr-1 or Ly-6G/Ly-6C, clone RB6-8C5), or isotype control antibodies (clones A19-3, R35-95, A95-1) (BD Biosciences). Following incubation for 20 min at 4°C, flourochrome-conjugated streptavidin, anti-mouse c-kit or CD117 (clone 2B8), anti-mouse Sca-1 or Ly-6A/E (clone D7), anti-mouse VEGFR2 (CD309, Clone Avas 12α1) (BD Biosciences) anti-mouse CD133 (clone 13A4, eBiosciences, San Diego, CA) or the matched isotype control antibodies were added into the incubation system at the final concentration of 10 µg/mL for each agent. Samples were further incubated in the dark for 20 min at 4°C. Antibody-stained cells were then washed with cold PBS containing 2% BSA. For measuring cell BrdU incorporation, cells were further processed using a BD BrdU Flow Kit (BD Biosciences) with the procedure provided by the manufacturer. For measuring cell expression of cyclin D1 and SP1, cells were further processed to make both cell membrane and nuclear membrane permeable for antibody using the procedure (without the step of DNA digestion with DNase) provided by BD BrdU Flow Kit (BD Biosciences). Permeabilized cells were incubated with 10 µg/mL of fluorochrome-conjugated anti-human/mouse cyclin D1 antibody (Clone DCS-6, Thermo Fisher Scientific, Inc.) or anti-human/mouse SP1 antibody (clone E-3, Santa Cruz Biotechnology, Inc.) in the dark for 20 min at room temperature. To cell samples incubated with anti-human/mouse SP1 antibody,  flourochrome-conjugated anti-mouse IgG2a, (10 µg/mL, clone R-1915, BD Biosciences) was then added. The cells were further incubated in the dark for 20 min at room temperature. The background staining control samples were incubated with the fluorochrome-conjugated 2nd antibody only. At the end of the staining procedure, cells were washed with the washing buffer provided with the BD BrdU Flow Kit (BD Biosciences) and then suspended in 0.5 ml of PBS containing 1% paraformaldehyde. Analysis of cell phenotypes, expression of SP1, and BrdU incorporation as well as sorting of selected cell subpopulations were performed on a FACSAria Fusion flow cytometer with FACSDiva software (Becton Dickinson, San Jose, CA). Cell populations of interest were gated based on their marker or marker combinations. Depending on the cell types analyzed, the number of cells acquired in each sample was in the range of 200,000 to 300,000. <strong><em>Co-immunoprecipitation and mass spectrometry</em></strong> Nucleated BMCs were isolated from Sca-1 KO and wildtype C57BL/6 mice following 24 h systemic <em>E. coli</em> infection. Isolated BMCs were lysed with a lysing buffer (10 mM Tris-HCl buffer containing 1% Triton X-100, 5 mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 1 mM PMSF, 50 mM sodium fluoride, 5 mg/ml aprotinin, 5 mg/ml pepstatin, and 5 mg/ml leupeptin, pH 7.6) for 30 min. After removing debris in cell lysates by centrifugation at 10,000 g for 5 min at 4°C, soluble protein samples in the supernatants were collected. Co-immunoprecipitation of Sca-1-associated proteins was performed using rat anti-mouse Sca-1 monoclonal antibody (clone E13-161.7, BD Biosciences) and Pierce® Co-Immunoprecipitation (Co-IP) Kit (Thermo Scientific, Frederic, MD). Sca-1 co-immunoprecipitated protein fractions were resolved using the 12% SDS-PAGE ready gel (Bio-Rad Laboratories, Hercules, CA). Recovery of the specific 21 KDa bands identified with Sca-1 pulldown in the SDS-PAGE gel was processed according to the typical in-gel digestion. Tryptic peptides were analyzed by Thermo Scientific Orbitrap Elite mass spectrometer coupled with Easy nano-LC system (Waltham, MA).  The protein matches were identified using Thermo Proteome Discoverer 1.4 with the Mascot search program (Matrix Science, London, UK) against the Mouse of SwissProt database.  A Percolator was used for the validation of peptide matches. <strong><em>Immunohistochemistry and morphological analysis</em></strong> Sorted marrow lin<sup>-</sup>ckit<sup>+</sup> cells from Sca-1 KO and wildtype C57BL/6 mice following 24 h systemic <em>E. coli</em> infection were fixed with 8% paraformaldehyde in PBS for 30 min. After washing 3 times with PBS containing 1% BSA, fixed cells were smeared onto poly-L-lysine-coated glass slides (Sigma-Aldrich) followed by a brief air dry. Fixed cells on slides were blocked with 10% goat serum (BioLegend) for 1 hour. After cell surface staining of Sca-1 with fluorochrome-conjugated rat anti-mouse Sca-1 (clone D7, BD Biosciences) for 1 h, the cell smears were washed 3 times with PBS containing 1% BSA. Cells were permeabilized with 0.5% Tween-20 (Sigma-Aldrich) in PBS for 10 min. After washing 3 times with 0.1% Tween-20, cells on slides were sequentially re-blocked with 10% goat serum, stained with fluorochrome-conjugated rabbit polyclonal antibody against mouse ras-related C3 botulinum toxin substrate 2 (Rac2, Bioss Inc., Woburn, MA), and eventually mounted with coverslips using the mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI, Abcam, Cambridge, United Kingdom) for imaging analysis under the Olympus IX81 time-lapse deconvolution fluorescent/phase contrast microscope (Olympus America Inc.). Subcutaneously implanted Matrigel plug samples collected from mice were fixed with 10% paraformaldehyde in PBS for 24 h. After washing 3 times with PBS, the fixed samples were processed using Leica ASP300S Fully Enclosed Tissue Processor (Leica Biosystems, Deer Park, IL) and paraffin-embedded with Leica Histore Arcadia H and C (Leica Biosystems). Microtome sections (5-10 µM) of paraffin-embedded tissue blocks were prepared using Leica RM2235 (Leica Biosystems). Sections were mounted on Superfrost Plus Microscope Slides (Fisher Healthcare, Pittsburgh, PA) and sequentially subjected to steps of deparaffinization, hydration, antigen retrieval, and blocking with 0.1% BSA. Slides were stained with fluorochrome-conjugated rabbit polyclone antibody against mouse von Willebrand factor (vWF, Bioss Inc.) Stained slides were mounted with coverslips using the Vectashield Antifade Mounting Medium with DAPI (Vector Laboratories, Inc. Newark, CA). Lung Tissue samples collected from mice were fixed with 4% paraformaldehyde at 4°C for 24 h. After washing 3 times in PBS, lung tissue samples were sequentially placed into 15% sucrose solution in PBS for 2 h at 4°C and 30% sucrose solution in PBS for 24 h at 4°C. Tissue samples were then frozen with optimal cutting temperature compound (OCT) (Sakura Tissue-Tek, Torrance, CA). Cryostat sections (10 µM) of OCT-embedded tissue were prepared using Leica CM1950 (Leica Biosystems). Sections were mounted on Superfrost Plus Microscope Slides (Fisher Healthcare), permeabilized with 0.2% Triton (Trition X-100 Sigma-Aldrich), and blocked with Normal Serum Block (Biolegend). Slides were stained with fluorochrome-conjugated Lycopresision Esculentum Lectin (isolectin B4, Thermo Fisher Scientific Inc.). Stained slides were mounted with coverslips using Vectashield Antifade Mounting Medium with DAPI (Vector Laboratories, Inc.) for imaging analysis under an Olympus FV1000 IX81 Spectral Confocal Microscope (Olympus America Inc.). <strong><em>Real-time RT-PCR</em></strong> Total RNA samples were prepared from marrow lin<sup>-</sup>c-kit<sup>+</sup>Sca-1<sup>-</sup> and lin<sup>-</sup>c-kit<sup>+</sup> cells with RNeasy Plus Mini Kit (Qiagen, Valencia, CA). Real-time RT-PCR analysis of mRNA expression by cells was performed as reported previously (24). Each RNA sample was subjected to 2-step real-time RT-PCR using iScriptTM Reverse Transcription Supermix kit and SsoFastTM EvaGreen® Supermix kit (Bio-Rad Laboratories), respectively, on the CFX96TM Real-Time System (Bio-Rad Laboratories). The amplification primer pairs were:  Sca-1:            Forward           5’-GTTTGCTGATTCTTCTTGTGGCCC                       Reverse           5’-ACTGCTGCCTCCTGAGTAACAC VEGFR2:       Forward           5’-ATCCAGATGAGGGCAAGTTTAG                        Reverse           5’-GCATACCCACTGACTGTGATAG 18SrRNA:       Forward           5’-ATTCGAACGTCTGCCCTATAA                        Reverse           5’-GTCACCCGTGGTCACCATG These sets of primers were designed using Primer Express software (Life Technologies Co, Carlsbad, CA). The expression of Sca-1 and VEGFR2 mRNA was determined by normalizing the cycle threshold (CT) number of their mRNA with that of 18S rRNA in each sample. Changes in specific gene mRNA expression by cells from groups with different treatments are expressed as fold alterations over the baseline expression by cells from the corresponding control group. <strong><em>ELISA determination</em></strong> Lung tissue homogenate (10%) was prepared by homogenizing lung tissue in PBS containing cOmplete Tablets Mini protease inhibitor cocktail (1 tablet/10 ml of PBS, Sigma-Aldrich) with a TH-115 homogenizer (OMNI International, Kennesaw GA). After centrifugation at 10000 g for 10 min, the supernatant of each tissue homogenate sample was collated. VEGFR2, SDF-1, and IFN-γ concentrations in plasma and lung tissue homogenate samples were determined with Quantikine Mouse VEGF Immunoassay kit, Mouse CXCL12/SDF-1 Immunoassay Kit, and Mouse IFN-γ Immunoassay Kit (R&D Systems, Minneapolis, MN), respectively. G-CSF levels in plasma and lung tissue homogenate samples were determined with the Mouse CSF3 (G-CSF) ELISA Kit (Thermo Scientific). Protein content in lung tissue homogenate samples was determined using the BCA Protein Assay Kit (Thermo Fisher Scientific, Inc.) with the protocol provided by the manufacturer. <strong><em>Statistical analysis</em></strong> Data were presented as mean±SEM. The sample size is indicated in each figure legend. Statistical analysis was conducted using Student’s t-test, one-way ANOVA followed by Student-Newman-Keuls test, liner correlation, and Chi-square test. Difference with statistical significance is accepted at p &lt; 0.05.
description: # The bone marrow endothelial progenitor cell response to septic
 infection: Dataset ## Author Information 1. A. Principal Investigator
 Contact Information Name: Ping Zhang Institution: Northeast Ohio Medical
 University Address: Rootstown, OH USA Email:
 [pzhang@neomed.edu](mailto:pzhang@neomed.edu) B. Associate or
 Co-investigator Contact Information Name: Xin Shi Institution: Northeast
 Ohio Medical University Address: Rootstown, OH USA Email:
 [xshi@neomed.edu](mailto:xshi@neomed.edu) Name: Kevin J. Simms
 Institution: Northeast Ohio Medical University Address: Rootstown, OH USA
 Email: [ksimms@neomed.edu](mailto:ksimms@neomed.edu) Name: Thomas J. Ewing
 Institution: West Clinical Laboratory, Lakeland Regional Health Medical
 Center Address: Lakeland, FL USA
 Email:[tewing5@kent.edu](mailto:tewing5@kent.edu) Name: Yuan-Ping Lin
 Institution: 3R Life Sciences Ltd. Address: Kaohsiung City, Taiwan Email:
 [benyplin.3rtwn@gmail.com](mailto:benyplin.3rtwn@gmail.com) Name: Yi-Ling
 Chen Institution: Institute for Translational Research in Biomedicine
 Address: Kaohsiung City, Taiwan Email:
 [rylchen.msu@gmail.com](mailto:rylchen.msu@gmail.com) Name: John N. Melvan
 Institution: Memorial Cardiac and Vascular Institute, Memorial Healthcare
 System Address: Hollywood, FL USA
 Email:[jmelvan@mhs.net](mailto:jmelvan@mhs.net) Name: Robert W. Siggins
 Institution: Louisiana State University Address: New Orleans, LA USA
 Email: [rsiggi@lsuhsc.edu](mailto:rsiggi@lsuhsc.edu) 2. Duration of work
 on manuscript: 2008-2023 ## SHARING/ACCESS INFORMATION NA ## DATA &
 FILE OVERVIEW Files are organized by figures in the manuscript and are
 composed of the corresponding data for each figure. A) Fig 1 B) Fig 2 C)
 Fig 3 D) Fig 4 E) Fig 5 F) Fig 6 G) Fig 7 H) Fig 8 I) Fig 9 Definition of
 terms:"lin-" = "lineage negative", "C+" =
 "ckit positive", "V" = "VEGFR2 positive",
 "V-" = "VEGFR2 negative", "LKS" =
 "lineage negative, ckit positive, Sca-1 positive cells",
 "Br+" = "BrdU positive", "Br-" = "BrdU
 Negative", "MCF" = "mean channel flourescence
 intensity", "WT" = "Wildtype", "SP1+" =
 "SP1 Positive", "EPC" = "Endothelial Progenitor
 Cells". *E.coli* injection doses are in CFUs. ### DATA-SPECIFIC
 INFORMATION FOR: Fig 1 1. Representative plots of flow cytometry for
 analyzing changes in bone marrow LKS, LKS VEGFR2+, and LKS CD133+VEGFR2+
 cell subtypes in mice 12 and 24 h following i.v. injection with saline
 (control) or 1 × 10^8 *E. coli*. A) The folder labeled "12h"
 consists of flow cytometry plots of bone marrow LKS cells harvested 12h
 after beginning the experiment. "E7", "E8", and
 "S" denote the number assigned to the mouse. And if the mouse is
 part of the control group (S) or experimental group (E). B) The folder
 labeled "24h" consists of flow cytometry plots of bone marrow
 LKS cells harvested 24h after beginning the experiment. "E" =
 i.v. injected with *E.coli* and "S" = i.v. injected with saline
 C) The folder labeled "ISO" consists of flow cytometry plots of
 bone marrow LKS cells labeled with isotype control antibody. D) Loose
 files: "Scan_0040.pdf" and "Scan_20200308 (2-6).png"
 are compilations of Flow cytometry plots found in folders "12h",
 and "24h". ### DATA-SPECIFIC INFORMATION FOR: Fig 2 1. Changes
 in bone marrow LKS cell populations as well as Sca-1 expression as
 reflected by mean channel fluorescence intensity (MCF) by LKS cells
 following septic infection. A) "BMC phenotyping data.xlsx" = a
 compilation of flow cytometry data found in files
 "R01-EPC-2(B-F)" showing the changes in expression of
 "LKS", "VEGFR2", "CD133"
 "lineage", and "ckit" in repeated experiments where
 mice are i.v. injected with saline, 1x10^7 *E.coli*, and 1x10^8 *E.coli*.
 The repeat experiments ended at 12h, 24h, 48h, 72h, and 7 days after i.v.
 injection. ### DATA-SPECIFIC INFORMATION FOR: Fig 3 1. Sca-1 protein
 expression by bone marrow stem/progenitor cell types 24 h following
 bacteremia. A) The folder labeled "Fig-3A" contains data
 previously explained in "Fig 2" B) The folder labeled
 "Fig-3B": "HSC-BL6-8 data.xls" shows the fold change
 in Sca-1 expression in mouse bone-marrow cells after i.v. injection of
 saline, 1x10^6 *E.coli*, or 1x10^8 *E.coli*. obtained by RT-PCR. File
 "HSC-BL6-8 results.xls" contains the RT-PCR data for the gene
 expression of Sca-1, GCSFr, Pu.1, and Gata3 genes in mice i.v. injected
 with 1x10^6 *E.coli*, 1x10^8 *E.coli*, or saline. ### DATA-SPECIFIC
 INFORMATION FOR: Fig 4 1. Fold changes in bone marrow BrdU+ and BrdU− LKS
 VEGFR2+ cells in mice 24 h following i.v. injection with 1 × 10^8 *E.
 coli*. Upregulation of cyclin D1 expression by marrow lin−c-kit+ cells 24
 h following i.v. injection with 5 × 10^7. *E. coli*. Increase in SP1+
 cells in bone marrow LKS and lin−c-kit+ cell subpopulations 18h post i.v.
 injection with 5 × 10^7 *E. coli*. Representative images of 5-week
 vasculogenic activity in implanted matrigel plugs containing bone marrow
 lin−c-kit+ cells from donor GFP+ mice with i.v. injected saline and
 heat-inactivated *E. coli* 1 × 10^8, respectively, for 24h. A) The folder
 labeled "Fig-4A" contains flow cytometry data of LKS cells that
 are BrdU+ vs. BrdU-, when i.v. injected with 1x10^8 *E.coli* for 24h. B)
 The folder labeled "Fig-4B C" contains flow cytometry data
 showing the percent amount and MCF of cyclinD1 in LKS cells of GBP7KO mice
 and WT mice after i.v. injection of 5x10^7 *E.coli* for 24h. C) The folder
 labeled "Fig-4D" contains flow cytometry data showing the
 percent amount and MCF of SP1 in LKS cells of mice i.v. injected with
 5x10^7 *E.coli* for 24. D) The folder labeled "Fig-4E1-3"
 contains confocal microscope images at 50X and 400X magnification of
 5-week vasculogenic activity in implanted matrigel plugs containing bone
 marrow lin−c-kit+ cells from donor GFP+ mice injected with i.v. saline and
 heat-inactivated 1 × 10^8 *E. coli* for 24 h. ### DATA-SPECIFIC
 INFORMATION FOR: Fig 5 1. Representative flow cytometry plots of
 phenotypic conversion to c-kit+Sca-1+ cells and increase in
 VEGFR2-expressing c-kit+Sca-1+ cells in cultured marrow lin−c-kit+Sca-1−
 24 h following LPS stimulation. Upregulation of Sca-1 mRNA expression by
 cultured bone marrow lin−ckit+ Sca-1− cells 24 h following LPS
 stimulation. Changes in cell subpopulations in cultured marrow
 lin−c-kit+Sca-1− 24 h following LPS stimulation. A) The folder labeled
 "Fig 5A C D" contains flow cytometry data and plots of the
 phenotypic conversion of LKS cells after being cultured with LPS for 24h.
 B) The folder labeled "Fig 5B" contains RT-PCR data showing the
 fold change of gene expression for Sca-1, GCSFr, and Pu.1 for LKS cells
 cultured with and without LPS. ### DATA-SPECIFIC INFORMATION FOR: Fig 6 1.
 Changes in cell subtypes in cultured bone marrow lin−c-kit+Sca-1− cells 24
 h following LPS + VEGF stimulation. Representative flow cytometry plots of
 increase in BrdU incorporation into c-kit+Sca-1+VEGFR2+ cell subtype in
 cultured bone marrow lin−c-kit+Sca-1− cells 24 h following LPS + VEGF
 stimulation. Sca-1 expression by different cell subtypes in bone marrow
 lin− cells cultured for 24 h in the absence and presence of LPS + VEGF
 stimulation. The significant correlation between Sca-1 expression and BrdU
 incorporation in bone marrow lin−c-kit+Sca-1− cells cultured for 24 h. A)
 The folder labeled "Fig 6A B C G" contains flow cytometry data
 showing the change in gene expression in LKS cells with stimulation from
 LPS and VEGF. B) The folder labeled "Fig 6D EPC-Bl6-3c Images"
 contains flow cytometry plots. C) The folder labeled "Fig 6E F"
 contains flow cytometry showing the incorporation of BrdU in LKS cells
 after being cultured with and without LPS. ### DATA-SPECIFIC INFORMATION
 FOR: Fig 7 1. Changes in plasma and lung tissue cytokine levels following
 i.v. injection with 1 × 10^8 *E. coli*. A) Contains data from multiple
 ELISA protein assays of mouse plasma and lung tissue showing changes in
 the amount of cytokines VEGF, G-CSF, SDF1, and IFN-γ. ### DATA-SPECIFIC
 INFORMATION FOR: Fig 8 1. Increase in circulating EPCs in mice 48 h
 following i.v. injection with 1 × 10^8 *E. coli*. Changes in bone marrow
 cell recruitment into the lungs in mice with i.v. injected *E. coli* 1 ×
 10^8. A) The folder labeled "Fig 8A" contains flow cytometry
 data showing the cell counts of different bone marrow cell subtypes. B)
 The folder labeled "Fig 8B1-5" contains the confocal imaging
 data of mice that were recipients of donor EGFP+ bone marrow cells. ###
 DATA-SPECIFIC INFORMATION FOR: Fig 9 1. SDS-PAGE (silver stain) exhibition
 of a 21-kDa protein fraction co-immunoprecipitated with Sca-1 in nucleated
 bone marrow cell lysates from mice 24 h following i.v. injection of 1x10^7
 *E.coli*. NSPP, non-specific pulldown fraction. Mass spectrometry
 identified enrichment of Rac2 in this 21-kDa protein fraction.
 Co-localized surface expression of Sca-1 and Rac2 by marrow precursor
 cells in mice 24 h following i.v. injection 1x10^7 *E.coli*. Fold changes
 in VEGFR2 mRNA expression by mouse bone marrow lin−ckit+ cells cultured
 for 24 h in the absence and presence of LPS plus Sca-1 crosslinking
 antibodies. Representative image of late EPC colony in matrigel culture of
 mouse bone marrow lin−c-kit+ cells for 8 weeks and changes in late EPC
 colonies in cultured bone marrow lin−ckit+ cells from wild-type and Sca-1
 KO mice 24 h following i.v. challenge with heat-inactivated 1 × 10^8 *E
 coli*. A) The folder labeled "Fig 9A B" contains co-ip data B)
 The folder labeled "Fig 9C" contains RT-PCR data showing the
 fold change in lin-ckit+ cells cultured with and without LPs and Sca-1
 crosslinking anti-bodies C) The folder labeled "Fig 9D" contains
 the colony counts and images of EPC from WT and Sca-1 KO mice. D) The
 folder labeled "Fig 9E" contains BCA protein assay data showing
 the amount of wet weight/mg protein of mouse lungs.