(A) Schematic strategy for MK preparation, scRNA-seq and data analysis. (B) Clustering of 1712 bone marrow MKs. (C) Heatmap of signature gene expression in MK subpopulations (fold-change >1.5, p …
Signature genes of MK1 to MK5.
The means and p value of the average expression level of interacting molecule 1 in cluster 1 and interacting molecule 2 in cluster 2 by CellPhoneDB.
(A) Flow cytometry gating for isolation of MKs (CD41+ FSChigh) in bone marrow for scRNA-seq. (B) Quality control of scRNA-seq data. Violin plots showing the number of unique genes (gene number) …
(A) Radar charts showing cell similarities of our single cell dataset with the published bone marrow MK single cells (Pariser et al., 2021; Yeung et al., 2020), bone marrow immune cells and myeloid …
(A) Re-clustering of 1712 MKs from 5368 cells. (B) Violin plots showing MK marker gene expression (Aburima et al., 2021; Bernardes et al., 2020; Kanaji et al., 2005; Liu et al., 2021; Couldwell and …
(A) Volcano plot showing MK5 and MK1 to 4 enriched genes (|fold change|>1.4, p value <0.05). (B–C) Gene Ontology (GO) analysis showing MK5 enriched immune pathway genes and MK1 to 4 mainly enriched …
(A) Dotplots of significant cytokine ligand (source) -receptor (target) interactions between MKs and immune cells discovered using CellChat. Color indicates communication probabilities, and bubble …
(A) Representative flow cytometry plots of gating strategy of CD41 and CXCR4 in the bone marrow of control mice and mice 3 days after L. monocytogenes infection. (B) Relative expression of Pf4, Vwf …
(A) Distribution of myeloid cells to CXCR4low or CXCR4high MKs 3 days after L. monocytogenes infection. Representative images of MKs (blue), CXCR4 (red), and myeloid cells (green) in mouse bone …
Distance of actual myeloid cells to the closest CXCR4low and CXR4high MKs 3 days after L. monocytogenes infection.
Distance of randomly positioned myeloid cells to the closest CXCR4low and CXR4high MKs 3 days after L. monocytogenes infection.
Mean distance of randomly positioned myeloid cells to the closest CXCR4low and CXR4high MKs 3 days after L. monocytogenes infection from 500 times simulations.
(A–B) Representative immunofluorescent staining image (A) and statistical analysis (B) showing association of MKs (blue) and myeloid cells (green) in the bone marrow of control mice and mice 3 days …
(A–B) Quantification of phagocytosis abilities in neutrophils (A) and macrophages (B) without or with CXCR4low MKs or CXCR4high MKs as indicated. (C–E) TNFα and IL-6 protein levels in CXCR4low and …
(A) Schema for diphtheria toxin (DT) and L. monocytogenes administration used for the experiments shown in (B–E). (B) Representative images of MKs (blue, indicated by arrows) and vascular …
(A) Schema for diphtheria toxin (DT) and L. monocytogenes administration used for the experiments shown in (B–D). (B) Number of bone marrow hematopoietic stem and progenitor cells in Pf4Cre+; Rosa26f…
The LacZ activity in B3Z T cells with or without co-culture as indicated. Ctrl indicates B3Z cells without MKs co-culture; bone-marrow-derived DCs, CXCR4low MKs and CXCR4high MKs were pulsed with …
(A) Representative image of CD41 (blue), CD150 (red), EMCN (green), and lineage cells (white) in bone marrow from control mice or mice at 3 days after L. monocytogenes infection. dHSC and dEC …
Distance of HSCs to MKs in the bone marrow from control mice or mice 3 days after L. monocytogenes infection.
(A) Bone marrow MKs (blue) and sinusoids (green) in control mice and mice 3 days after L. monocytogenes infection. CD41, blue; EMCN, green. Yellow arrows indicate MKs egressed into sinusoids. ‘S’ …
(A) Schematic depicting the strategy of scRNA-seq using bone marrow MKs from mice 3 days after L. monocytogenes infection. (B) Violin plots showing the number of unique genes (gene number) before …
(A–H) Relative expression of Cxcl12 (A–D) and frequency of CXCL12DsRed cells (E–H) in the bone marrow (A), lung (B), liver (C), and spleen (D) from control mice and mice 3 days after L. monocytogenes…
(A–B) Fraction of BrdU+ and Annexin V+ MKs in CXCR4high MKs in the liver (A) and spleen (B) from control mice or mice 3 days after L. monocytogenes infection. (C) Quantification of CXCR4low MKs in …
(A) Gene set variation analysis (GSVA) of each MK subpopulation under control or L. monocytogenes infection, colored by row-scaled GSVA enrichment scores. (B) Antigen processing and presentation …
(A) Schema for transfer experiments using tdTomato+ MKs from Pf4Cre+; Rosa26fs-tdTomato+/- mice into recipients 1 day following L. monocytogenes infection. (B) Transfused MKs (red) and sinusoids …
MK (red, arrowhead) migrating into the bone marrow sinusoids (green, labeled by Dextran-FITC) by live imaging in the bone marrow of Pf4Cre+; Rosa26fs-tdTomato+/-mice 24 hr after L. monocytogenes …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Antibody | Anti-CD41a (mouse monoclonal) | eBioscience | Cat#17-0411-82 RRID:AB_1603237 | FACS (1 μl per test) |
Antibody | Anti-CXCR4 (mouse monoclonal) | eBioscience | Cat#53-9991-80 RRID:AB_953573 | FACS (1 μl per test) |
Antibody | Anti-CD11b (mouse monoclonal) | eBioscience | Cat#12-0112-82 RRID:AB_2734869 | FACS (1 μl per test) |
Antibody | Anti-F4/80 (mouse monoclonal) | eBioscience | Cat#17-4801-80 RRID:AB_2784647 | FACS (1 μl per test) |
Antibody | Anti-Gr-1 (mouse monoclonal) | Biolegend | Cat#108424 RRID:AB_2137485 | FACS (1 μl per test) |
Antibody | Anti-Ly-6C (mouse monoclonal) | Biolegend | Cat#128022 RRID:AB_10639728 | FACS (1 μl per test) |
Antibody | Anti-CD11c (mouse monoclonal) | eBioscience | Cat#12-0114-82 RRID:AB_465552 | FACS (1 μl per test) |
Antibody | Anti-CD45.1 (mouse monoclonal) | eBioscience | Cat#15-0453-82 RRID:AB_468759 | FACS (1 μl per test) |
Antibody | Anti-CD45.2 (mouse monoclonal) | Biolegend | Cat#109831 RRID:AB_10900256 | FACS (1 μl per test) |
Antibody | Anti-CD4 (mouse monoclonal) | eBioscience | Cat#12-0041-82 RRID:AB_465506 | FACS (1 μl per test) |
Antibody | Anti-CD8a (mouse monoclonal) | Biolegend | Cat#100707 RRID:AB_312746 | FACS (1 μl per test) |
Antibody | Anti-IFN-γ (mouse monoclonal) | Biolegend | Cat#505813 RRID:AB_493312 | FACS (1 μl per test) |
Antibody | Anti-IL-4 (mouse monoclonal) | Biolegend | Cat#504118 RRID:AB_10898116 | FACS (1 μl per test) |
Antibody | Anti-CD34 (mouse monoclonal) | eBioscience | Cat#11-0341-82 RRID:AB_465021 | FACS (1 μl per test) |
Antibody | Anti-Sca-1 (mouse monoclonal) | Biolegend | Cat#108114 RRID:AB_493596 | FACS (1 μl per test) |
Antibody | Anti-c-Kit (mouse monoclonal) | Biolegend | Cat#105812 RRID:AB_313221 | FACS (1 μl per test) |
Antibody | Anti-CD135 (mouse monoclonal) | Biolegend | Cat#135314 RRID:AB_2562339 | FACS (1 μl per test) |
Antibody | Anti-CD3ε (mouse monoclonal) | Biolegend | Cat#100310 RRID:AB_312675 | FACS (1 μl per test) |
Antibody | Anti-B220 (mouse monoclonal) | Biolegend | Cat#103210 RRID:AB_312995 | FACS (1 μl per test) |
Antibody | Anti-TER-119 (mouse monoclonal) | Biolegend | Cat#116210 RRID:AB_313711 | FACS (1 μl per test) |
Antibody | Anti-IgM (mouse monoclonal) | eBioscience | Cat#15-5790-82 RRID:AB_494222 | FACS (1 μl per test) |
Antibody | Anti-CD16/32 (mouse monoclonal) | Biolegend | Cat#101333 RRID:AB_2563692 | FACS (1 μl per test) |
Antibody | Anti-CD127 (mouse monoclonal) | Biolegend | Cat#135021 RRID:AB_1937274 | FACS (1 μl per test) |
Antibody | Anti-TNFα (mouse monoclonal) | Invitrogen | Cat#17-7321-81 RRID:AB_469507 | FACS (1 μl per test) IF (1:100) |
Antibody | Anti-IL-6 (mouse monoclonal) | Biolegend | Cat#504507 RRID:AB_10694868 | FACS (1 μl per test) IF (1:100) |
Antibody | Anti-BrdU (mouse monoclonal) | eBioscience | Cat#11-5071-42 RRID:AB_11042627 | FACS (1 μl per test) |
Antibody | Anti-Endomucin (mouse polyclonal) | R&D | Cat#AF4666 | IF (1:100) |
Antibody | Anti-CD150 (mouse monoclonal) | Biolegend | Cat#115908 RRID:AB_345278 | IF (1:100) |
Antibody | Anti-Lineage Panel (mouse monoclonal) | Biolegend | Cat#133307 RRID:AB_11124348 | IF (1:100) |
Antibody | Anti-Goat AF488 (goat polyclonal) | Invitrogen | Cat#A32814 RRID:AB_2762838 | IF (1:1000) |
Antibody | Anti-TNF-alpha (mouse monoclonal) | Sino Biological | Cat#50349-R023 | 2 μg ml–1 |
Antibody | Anti-Rabbit AF488 (rabbit polyclonal) | Invitrogen | Cat#R37118 RRID:AB_2556546 | IF (1:1000) |
Antibody | Anti-OVA257-264 (SIINFEKL) peptide bound to H-2Kb (mouse monoclonal) | Invitrogen | Cat#17-5743-82 RRID:AB_1311286 | FACS (1 μl per test) |
Antibody | Anti-IL-2 (mouse monoclonal) | eBioscience | Cat#25-7021-80 RRID:AB_1235007 | FACS (1 μl per test) |
Chemical compound, drug | Diphtheria toxin (DT) | Sigma-Aldrich | Cat#D0564-1MG | 40 μg kg–1 body mass |
Chemical compound, drug | BrdU (5-Bromo-2´-Deoxyuridine) | Sigma-Aldrich | Cat#B5002-250mg | 125 mg kg–1 body mass |
Chemical compound, drug | CFSE (5-Carboxyfluorescein, Succinimidyl Ester) | Invitrogen | Cat#C2210 | 2.5 μM |
Chemical compound, drug | GM-CSF | Abbkine | Cat#PRP2116 | 10 ng ml–1 |
Chemical compound, drug | IL-4 | novoprotein | Cat#CK15 | 10 ng ml–1 |
Chemical compound, drug | Tamoxifen | Sigma-Aldrich | Cat#T5648 | 20 mg ml–1 corn oil |
Commercial kit | Chromium Single Cell 3′ GEM, Library & Gel Bead Kit v3 | 10 x Genomics | PN-1000075 | |
Commercial kit | Chromium Chip B Single Cell Kit | 10 x Genomics | PN-1000074 | |
Cell line (Mus musculus) | NCTC clone 929 | ATCC | CCL-1 RRID:CVCL_0462 | |
Cell line (Mus musculus) | B3Z hybridoma CD8 T cell | Dr. Nilabh Shastri | ||
Other | scRNA sequencing data (raw and processed data) | This paper | GEO: GSE168224 | |
Genetic reagent (Mus musculus) | C57BL/6 J | Shanghai Model Organisms | ||
Genetic reagent (Mus musculus) | Tg(Pf4-icre)Q3Rsko/J (Pf4Cre) | Jackson Laboratory | Stock No: 008535 | |
Genetic reagent (Mus musculus) | Gt(ROSA) 26Sortm1(HBEGF) Awai/J (Rosa26fs-iDTR) | Jackson Laboratory | Stock No: 007900 | |
Genetic reagent (Mus musculus) | Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J (Rosa26fs-mTmG) | Jackson Laboratory | Stock No: 007576 | |
Genetic reagent (Mus musculus) | Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J (Rosa26fs-tdTomato) | Jackson Laboratory | Stock No: 007905 | |
Genetic reagent (Mus musculus) | SclCreER mice | Göthert et al., 2005 | ||
Genetic reagent (Mus musculus) | Cxcl12tm2.1Sjm/J (Cxcl12fs-DsRed) | Jackson Laboratory | Stock No: 022458 | |
Genetic reagent (Mus musculus) | C57BL/6-Tg(TcraTcrb)1,100Mjb/J (OT-I) | Jackson Laboratory | Stock No: 003831 | |
Strain, strain background (L. monocytogenes) | 10403 S | Bishop and Hinrichs, 1987 | ||
Software, algorithm | Cell ranger_3.0.2 | 10 x Genomics | tenx RRID:SCR_01695 | |
Software, algorithm | R_3.6.3 | https://cran.r-project.org/ | R 3.6.3 | |
Software, algorithm | Seurat_3.0.2 | Butler et al., 2018 | Seurat RRID:SCR_016341 | |
Software, algorithm | ggplot2_3.2.0 | https://cran.r-project.org/web/packages/ggplot2/index.html | ggplot2 RRID:SCR_014601 | |
Software, algorithm | clusterProfiler_3.12.0 | Yu et al., 2012 | clusterProfiler RRID:SCR_016884 | |
Software, algorithm | pheatmap_1.0.12 | https://cran.r-project.org/web/packages/pheatmap/ | pheatmap RRID:SCR_016418 | |
Software, algorithm | CellPhoneDB_2.1.7 | Efremova et al., 2020 | CellPhoneDB RRID:SCR_017054 | |
Software, algorithm | CellChat_1.1.3 | Jin et al., 2021 | CellChat 1.1.3 | |
Software, algorithm | symphony_1.0 | Kang et al., 2021 | symphony 1.0 | |
Software, algorithm | MetaNeighbor_1.10.0 | Crow et al., 2018 | MetaNeighbor RRID:SCR_016727 | |
Software, algorithm | iMAP_1.0.0 | Wang et al., 2021b | iMAP 1.0.0 | |
Software, algorithm | scmap_ 1.16.0 | Kiselev et al., 2018 | Scmap RRID:SCR_017338 | |
Software, algorithm | enrichplot_1.4.0 | Yu, 2019 | enrichplot 1.4.0 | |
Software, algorithm | Imaris_8.4 | Bitplane | Imaris RRID:SCR_007370 | |
Software, algorithm | FlowJo_10 | BD Bioscience | FlowJo RRID:SCR_008520 | |
Software, algorithm | ImageJ_ 1.8.0 | National Institutes of Health | ImageJ RRID:SCR_003070 | |
Other | DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) | Thermo Fisher | Cat#D1306 | IF (0.5 µg/mL) |
Other | Corn oil | Sigma-Aldrich | Cat#PHR2897 | Tamoxifen dissolution |
Other | Lymphocyte Separation Medium | TBD Science | Cat#LTS1077 | Liver cell isolation |