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Becton Dickinson facsaria ii
Induction of the EZH2 gene by Epstein-Barr virus (EBV) infection in primary B cells. (A) B cells isolated from peripheral blood mononuclear cells from a healthy donor were sorted using <t>FACSAria</t> II and infected or mock infected with WT EBV at a multiplicity of infection of ∼1. RNA was collected from the infected and mock-infected cells after 2 days. The mRNA was enriched, reverse transcribed, and subjected to RNA sequencing. Relative mRNA levels were calculated according to the frequency per kilobase of exon per million read values after normalization by the values of mock-infected sample. KMT, lysine methyltransferase; KDM, lysine demethylase. The RNA-seq data are available at the DDBJ Sequence Read Archive (accession ID DRA006767 ). (B and C) Peripheral B cells from different donors were infected with EBV as in panel A and analyzed by qRT-PCR. Relative EZH2 mRNA levels are shown after normalization with beta-2 microglobulin (B2M). Average and SD from three independent infections are shown. Student’s t test was performed. (D and E) Akata(−) cells were infected with EBV as in panel A and analyzed by qRT-PCR. Relative EZH1 and EZH2 mRNA levels are shown after normalization with beta-2 microglobulin (B2M). Average and SD from three independent infections are shown. Student’s t test was performed. *, P
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1) Product Images from "Regulation of Epstein-Barr Virus Life Cycle and Cell Proliferation by Histone H3K27 Methyltransferase EZH2 in Akata Cells"

Article Title: Regulation of Epstein-Barr Virus Life Cycle and Cell Proliferation by Histone H3K27 Methyltransferase EZH2 in Akata Cells

Journal: mSphere

doi: 10.1128/mSphere.00478-18

Induction of the EZH2 gene by Epstein-Barr virus (EBV) infection in primary B cells. (A) B cells isolated from peripheral blood mononuclear cells from a healthy donor were sorted using FACSAria II and infected or mock infected with WT EBV at a multiplicity of infection of ∼1. RNA was collected from the infected and mock-infected cells after 2 days. The mRNA was enriched, reverse transcribed, and subjected to RNA sequencing. Relative mRNA levels were calculated according to the frequency per kilobase of exon per million read values after normalization by the values of mock-infected sample. KMT, lysine methyltransferase; KDM, lysine demethylase. The RNA-seq data are available at the DDBJ Sequence Read Archive (accession ID DRA006767 ). (B and C) Peripheral B cells from different donors were infected with EBV as in panel A and analyzed by qRT-PCR. Relative EZH2 mRNA levels are shown after normalization with beta-2 microglobulin (B2M). Average and SD from three independent infections are shown. Student’s t test was performed. (D and E) Akata(−) cells were infected with EBV as in panel A and analyzed by qRT-PCR. Relative EZH1 and EZH2 mRNA levels are shown after normalization with beta-2 microglobulin (B2M). Average and SD from three independent infections are shown. Student’s t test was performed. *, P
Figure Legend Snippet: Induction of the EZH2 gene by Epstein-Barr virus (EBV) infection in primary B cells. (A) B cells isolated from peripheral blood mononuclear cells from a healthy donor were sorted using FACSAria II and infected or mock infected with WT EBV at a multiplicity of infection of ∼1. RNA was collected from the infected and mock-infected cells after 2 days. The mRNA was enriched, reverse transcribed, and subjected to RNA sequencing. Relative mRNA levels were calculated according to the frequency per kilobase of exon per million read values after normalization by the values of mock-infected sample. KMT, lysine methyltransferase; KDM, lysine demethylase. The RNA-seq data are available at the DDBJ Sequence Read Archive (accession ID DRA006767 ). (B and C) Peripheral B cells from different donors were infected with EBV as in panel A and analyzed by qRT-PCR. Relative EZH2 mRNA levels are shown after normalization with beta-2 microglobulin (B2M). Average and SD from three independent infections are shown. Student’s t test was performed. (D and E) Akata(−) cells were infected with EBV as in panel A and analyzed by qRT-PCR. Relative EZH1 and EZH2 mRNA levels are shown after normalization with beta-2 microglobulin (B2M). Average and SD from three independent infections are shown. Student’s t test was performed. *, P

Techniques Used: Infection, Isolation, RNA Sequencing Assay, Sequencing, Quantitative RT-PCR

Related Articles

other:

Article Title: In vitro Differentiation of Thymic Treg Cell Progenitors to Mature Thymic Treg Cells
Article Snippet: BD Biosciences FACSAria II (BD, model: FACSAria II).

Article Title: Sphingosine 1-phosphate receptor signaling establishes AP-1 gradients to allow for retinal endothelial cell specialization
Article Snippet: CD31+/CD45-/TER119- cells were sorted using BD FACSAria™ II (BD Biosciences).

Article Title: The landscape of accessible chromatin in quiescent and post-myocardial infarction cardiac fibroblasts
Article Snippet: Tcf21 lineage-traced eGFP+ cardiac fibroblasts were sorted on FACSaria II (BD Biosciences).

Purification:

Article Title: Complement receptor 3 mediates both sinking phagocytosis and phagocytic cup formation via distinct mechanisms
Article Snippet: RNA sequence analyses Mouse resident peritoneal cells were labeled with Alexa Fluor 488–conjugated anti-F4/80 antibodies, washed, and resuspended in autoMACS running buffer (Miltenyi Biotec), which contains PBS, 2-mM EDTA, 0.5% bovine serum albumin, and 0.09% sodium azide (pH 7.2). .. Purification of F4/80+ cells (mouse macrophages) was performed using a BD FACSAria II (or FACSAria III) cell sorter (BD Biosciences). .. The recovered suspension of F4/80+ cells was centrifuged at 300g for 5 min, and the supernatant was removed, followed by isolation of total RNA by solid-phase extraction using a Direct-zol RNA MicroPrep kit (Zymo Research), according to the manufacturer’s instructions.

Expressing:

Article Title: IL-21 from high-affinity CD4 T cells drives differentiation of brain-resident CD8 T cells during persistent viral infection
Article Snippet: 2D-Micropipette cell adhesion assay CD4 T cells from brains and spleens of IL21-VFP mice were enriched for using magnetic bead-based negative selection kits (STEMCELL Technologies Inc., Vancouver, BC). .. They were then sorted on a BD FACSAria II (BD Biosciences) based on expression of CD69 and VFP (IL21) to obtain a CD69+ VFP+ population and a CD69+ VFP− population. .. The relative 2D affinity of polyclonal I-Ab LT678 or VP1–122 specific CD4+ CD69+ T cells from either the brain or spleen of MuPyV infected mice was measured using the previously characterized 2D-MP ( , , ).

Cytometry:

Article Title: Functional variant analyses (FVAs) predict pathogenicity in the BRCA1 DNA double-strand break repair pathway.
Article Snippet: The quantified total p53, phosphop53 and phospho-p53/total p53 ratio were compared for the mutant, VUS and wild-type LCLs. .. Quantitative analysisFlow cytometry was performed using BD FACSAria II equipped with Blue (488 nm), Green (532 nm), Yellow (561 nm), Red (638 nm) and Violet lasers (407 nm). ..

FACS:

Article Title: Autophagy plays a protective role as an anti-oxidant system in human T cells and represents a novel strategy for induction of T-cell apoptosis.
Article Snippet: Flow cytometryFluorescence intensity was determined using BD LSR Fortessa (Becton Dickinson, Franklin Lakes, NJ, USA). .. Cell sorting was performed using BD FACSAria II (Becton Dickinson). .. Data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).

Staining:

Article Title: Micro-environmental sensing by bone marrow stroma identifies IL-6 and TGFβ1 as regulators of hematopoietic ageing
Article Snippet: B220+ cells were counted and stained with SAv-Brilliant Violet 605™ (BioLegend, 405229) and anti-Mouse IgM PE-Cyanine7 Antibody (Life Technologies, 25–5790–81). .. After wash, cells were stained with 7-Aminoactinomycin D (7AAD; Biotium 40037) and phenotypically defined early B precursors (7AAD− B220+ IgM−) sorted using a BD FACSAria II cell sorter (BD Biosciences). .. Analysis was performed using Flowjo software version 10.3 (Flowjo LLC, OR, USA).

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    Becton Dickinson bd facsaria ii
    Higher-Order Clustering is Inhibited by the Unliganded Extracellular Domain ( A ) Schematic illustration of possible mechanism underlying proteolytic activation of DR5. ( B )Comparing TRAIL sensitivity of the DR5-TEV-expressing BJAB cells to that of the DR5-WT-expressing BJAB cells. Caspase-8 activity was measured using the CaspGLOW red Caspase-8 activity kit (Biovision) and flow cytometry (BD <t>FACSAria</t> II) and was calculated as percentage of DsRed2+/GFP+ cells divided by percentage of GFP+ cells. Results were from 3 independent experiments (n=3), and expressed as mean ± SEM. ( C )TEV-induced apoptosis. DR5-deficient BJAB cells were transiently transfected with WT DR5-EGFP with (DR5-TEV) or without (DR5-WT) TEV cleavage site between the ECD and TMD. After transfection, BJAB cells were treated with TEV for 5 h before analysis. ( D )Examples of CFP and YFP photobleaching for WT DR5 without (left) and with (right) TRAIL. ( E )Receptor self-association of the WT DR5 and mutants. The effects of dimer-breaking mutation (G217Y) and trimer-breaking mutation (A222Y) with or without TRAIL pre-treatment are quantitated in the form of FRET efficiency. CFP/YFP co-transfected cells were used as FRET negative control. N.S indicates not significant. Twelve regions of interest (n=12) (ROI, e.g., plasma membrane bound DR5) from different cells were examined by FRET study. .
    Bd Facsaria Ii, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson cd74
    <t>CD74</t> −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p
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    Higher-Order Clustering is Inhibited by the Unliganded Extracellular Domain ( A ) Schematic illustration of possible mechanism underlying proteolytic activation of DR5. ( B )Comparing TRAIL sensitivity of the DR5-TEV-expressing BJAB cells to that of the DR5-WT-expressing BJAB cells. Caspase-8 activity was measured using the CaspGLOW red Caspase-8 activity kit (Biovision) and flow cytometry (BD FACSAria II) and was calculated as percentage of DsRed2+/GFP+ cells divided by percentage of GFP+ cells. Results were from 3 independent experiments (n=3), and expressed as mean ± SEM. ( C )TEV-induced apoptosis. DR5-deficient BJAB cells were transiently transfected with WT DR5-EGFP with (DR5-TEV) or without (DR5-WT) TEV cleavage site between the ECD and TMD. After transfection, BJAB cells were treated with TEV for 5 h before analysis. ( D )Examples of CFP and YFP photobleaching for WT DR5 without (left) and with (right) TRAIL. ( E )Receptor self-association of the WT DR5 and mutants. The effects of dimer-breaking mutation (G217Y) and trimer-breaking mutation (A222Y) with or without TRAIL pre-treatment are quantitated in the form of FRET efficiency. CFP/YFP co-transfected cells were used as FRET negative control. N.S indicates not significant. Twelve regions of interest (n=12) (ROI, e.g., plasma membrane bound DR5) from different cells were examined by FRET study. .

    Journal: Cell

    Article Title: Higher-Order Clustering of the Transmembrane Anchor of DR5 Drives Signaling

    doi: 10.1016/j.cell.2019.02.001

    Figure Lengend Snippet: Higher-Order Clustering is Inhibited by the Unliganded Extracellular Domain ( A ) Schematic illustration of possible mechanism underlying proteolytic activation of DR5. ( B )Comparing TRAIL sensitivity of the DR5-TEV-expressing BJAB cells to that of the DR5-WT-expressing BJAB cells. Caspase-8 activity was measured using the CaspGLOW red Caspase-8 activity kit (Biovision) and flow cytometry (BD FACSAria II) and was calculated as percentage of DsRed2+/GFP+ cells divided by percentage of GFP+ cells. Results were from 3 independent experiments (n=3), and expressed as mean ± SEM. ( C )TEV-induced apoptosis. DR5-deficient BJAB cells were transiently transfected with WT DR5-EGFP with (DR5-TEV) or without (DR5-WT) TEV cleavage site between the ECD and TMD. After transfection, BJAB cells were treated with TEV for 5 h before analysis. ( D )Examples of CFP and YFP photobleaching for WT DR5 without (left) and with (right) TRAIL. ( E )Receptor self-association of the WT DR5 and mutants. The effects of dimer-breaking mutation (G217Y) and trimer-breaking mutation (A222Y) with or without TRAIL pre-treatment are quantitated in the form of FRET efficiency. CFP/YFP co-transfected cells were used as FRET negative control. N.S indicates not significant. Twelve regions of interest (n=12) (ROI, e.g., plasma membrane bound DR5) from different cells were examined by FRET study. .

    Article Snippet: Flow cytometry was performed on BD FACSAria II.

    Techniques: Activation Assay, Expressing, Activity Assay, Flow Cytometry, Transfection, Mutagenesis, Negative Control

    Adjuvanticity of FlgE to Soluble Antigen in Mice. ( A–C ) Naïve OVA-specific T cells were purified from the spleens and inguinal lymph nodes of CD45.1 × OTII F1 mice by sorting using various antibodies to surface markers, labeled with eFluor450 and transferred into female C57BL/6 mice at 1 × 10 6 cells/mouse via tail vein injection. Twenty-four hours later, the recipients were treated or immunized via subcutaneous injection at the base of the tail with one of eight compositions, namely 1 μg OVA, 50 μg CpG-1826, 50 μg FlgE, 50 μg FlgEM each alone, or OVA plus the three stimulants individually. Control groups received 100 μL PBS. Three days later, the mice were sacrificed, and the draining inguinal lymph nodes were isolated and photographed ( A ). Single-cell suspension were made for staining with CD45.1 plus CD4 and then run on a BD FACSAria II and analyzed for eFluor450 intensity to quantify the proliferation of OVA-specific T cells ( B,C ). ( D ) To measure the humoral response, WT C57BL/6 mice were immunized with above 8 compositions except for increasing OVA doses to 100 μg. Two weeks later, the mice were sacrificed for serum harvest, and anti-OVA titers were measured using an ELISA as described in Materials and Methods. Data are representatives of two independent experiments that showed similar results. n = 3 mice for panel A–C, and n = 4 mice for panel D. *P

    Journal: Scientific Reports

    Article Title: Flagellar Hooks and Hook Protein FlgE Participate in Host Microbe Interactions at Immunological Level

    doi: 10.1038/s41598-017-01619-1

    Figure Lengend Snippet: Adjuvanticity of FlgE to Soluble Antigen in Mice. ( A–C ) Naïve OVA-specific T cells were purified from the spleens and inguinal lymph nodes of CD45.1 × OTII F1 mice by sorting using various antibodies to surface markers, labeled with eFluor450 and transferred into female C57BL/6 mice at 1 × 10 6 cells/mouse via tail vein injection. Twenty-four hours later, the recipients were treated or immunized via subcutaneous injection at the base of the tail with one of eight compositions, namely 1 μg OVA, 50 μg CpG-1826, 50 μg FlgE, 50 μg FlgEM each alone, or OVA plus the three stimulants individually. Control groups received 100 μL PBS. Three days later, the mice were sacrificed, and the draining inguinal lymph nodes were isolated and photographed ( A ). Single-cell suspension were made for staining with CD45.1 plus CD4 and then run on a BD FACSAria II and analyzed for eFluor450 intensity to quantify the proliferation of OVA-specific T cells ( B,C ). ( D ) To measure the humoral response, WT C57BL/6 mice were immunized with above 8 compositions except for increasing OVA doses to 100 μg. Two weeks later, the mice were sacrificed for serum harvest, and anti-OVA titers were measured using an ELISA as described in Materials and Methods. Data are representatives of two independent experiments that showed similar results. n = 3 mice for panel A–C, and n = 4 mice for panel D. *P

    Article Snippet: Cells were run on a BD FACSAria II and analyzed for eFluor450 intensity to reflect proliferation.

    Techniques: Mouse Assay, Purification, Labeling, Injection, Isolation, Staining, Enzyme-linked Immunosorbent Assay

    CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p

    Journal: PLoS Biology

    Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

    doi: 10.1371/journal.pbio.3001121

    Figure Lengend Snippet: CD74 −/− HSPCs show a higher potential to repopulate the BM. Lethally irradiated WT CD45.1 recipient mice were reconstituted with 7.5*10 4 sorted LSK cells from WT (CD45.1), and 7.5 × 10 4 sorted LSK from CD74 −/− (CD45.2) at a 1:1 ratio. Percent of donor-derived cells was analyzed in the BM after 6 and 18 weeks. (A) Total BM cells; Data A in S4 Data (B) myeloid cells; Data B in S4 Data (C) immature BM B cells; Data C in S4 Data and (D) mature BM B cells, Data D in S4 Data . (E) Percent of donor-derived cells was analyzed in LSK and CD34-LSK cells 18 weeks posttransplant. n = 6–8. Data E in S4 Data . n = 6–8. Bars show SEM. Unpaired two-tailed t test * p

    Article Snippet: Generation of chimeric mice For the microenvironment experiment: Lethally irradiated (950 Rad) C57BL/6 (WT) recipient mice were reconstituted with 5*106 WT or CD74−/− BM cells.

    Techniques: Irradiation, Mouse Assay, Derivative Assay, Two Tailed Test

    CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p

    Journal: PLoS Biology

    Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

    doi: 10.1371/journal.pbio.3001121

    Figure Lengend Snippet: CD74 −/− HSPCs demonstrate enhanced long-term self-renewal capacity. (A–F) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 3:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 3:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (A) Total BM cells; Data A in S5 Data (B) BM myeloid cells; Data B in S5 Data (C) BM immature B cells; Data C in S5 Data (D) LSK; Data D in S5 Data (E) CD34-/LSK; Data E in S5 Data (F) Mature BM B cells; Data F in S5 Data . n = 13. (G–L) Lethally irradiated WT(CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT(CD45.2) at a 9:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 9:1 ratio. Mice were analyzed 16 weeks after transplantation. Graphs show percent of donor-derived cells from both WTCD45.1/WTCD45.2 and WTCD45.1/CD74 −/− CD45.2 chimera. (G) Total BM cells; Data G in S5 Data (H) BM myeloid cells; Data H in S5 Data (I) BM immature B cells; Data I in S5 Data (J) LSK; Data J in S5 Data (K) CD34-/LSK; Data K in S5 Data (L) Mature BM B cells; Data L in S5 Data . n = 12. Bars show SEM. Unpaired two-tailed t test * p

    Article Snippet: Generation of chimeric mice For the microenvironment experiment: Lethally irradiated (950 Rad) C57BL/6 (WT) recipient mice were reconstituted with 5*106 WT or CD74−/− BM cells.

    Techniques: Irradiation, Mouse Assay, Derivative Assay, Transplantation Assay, Two Tailed Test

    Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p

    Journal: PLoS Biology

    Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

    doi: 10.1371/journal.pbio.3001121

    Figure Lengend Snippet: Expansion of HSPCs in the BM of CD74 −/− mice. (A) BM cells derived from WT or CD74 −/− were purified. Histograms show representative analysis of CD74 expression on HSPCs in WT and CD74 −/− mice. n = 3. (B) Total BM cellularity per femur and tibia in WT and CD74 −/− mice, Data A in S1 Data . (C–J) The percent of the different populations in WT and CD74 −/− -derived BM cells. (C) Lin-; Data B in S1 Data (D) Representative FACS analysis of WT and CD74 −/− HSPCs; (E) LSK; Data C in S1 Data (F) CD34-/LSK; Data D in S1 Data and (G) CD34+; Data E in S1 Data (H) CD150+CD48-LSK; Data F in S1 Data (I) CD150-CD48-/LSK; Data G in S1 Data and (J) CD150-CD48+/LSK; n = 14–18, Data H in S1 Data . (K) CFUC assay: Total BM cells from WT and CD74 −/− mice were seeded at 15,000 cells/mL in semisolid cultures supplemented with cytokines and nutrients. CFU-C were counted 7 days later; n = 7, Data I in S1 Data . Bars show SEM. Unpaired two-tailed t test * p

    Article Snippet: Generation of chimeric mice For the microenvironment experiment: Lethally irradiated (950 Rad) C57BL/6 (WT) recipient mice were reconstituted with 5*106 WT or CD74−/− BM cells.

    Techniques: Mouse Assay, Derivative Assay, Purification, Expressing, FACS, Two Tailed Test

    CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p

    Journal: PLoS Biology

    Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

    doi: 10.1371/journal.pbio.3001121

    Figure Lengend Snippet: CD74 −/− HSPCs have an advantage in BM repopulation. Lethally irradiated WT (CD45.1) mice were transplanted with BM derived from WT (CD45.1) and WT (CD45.2) at a 1:1 ratio, or BM derived from WT (CD45.1) and CD74 −/− (CD45.2) mice at a 1:1 ratio. (A) Representative BM FACS staining. Percent of donor-derived cells was analyzed in the BM after 6, 16, and 24 weeks in (B) Total BM cells; Data A in S3 Data (C) myeloid cells (CD11B+); Data B in S3 Data (D) B cells (B220+); Data C in S3 Data (E) LSK; Data D in S3 Data (F) CD34-/LSK; Data E in S3 Data (G) immature BM B cells (B220+IgD-); Data F in S3 Data (H) mature BM B cells (B220+ IgM+ IgD+); Data G in S3 Data . n = 8–18. Bars show SEM. Unpaired two-tailed t test * p

    Article Snippet: Generation of chimeric mice For the microenvironment experiment: Lethally irradiated (950 Rad) C57BL/6 (WT) recipient mice were reconstituted with 5*106 WT or CD74−/− BM cells.

    Techniques: Irradiation, Mouse Assay, Derivative Assay, FACS, Staining, Two Tailed Test

    CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *

    Journal: PLoS Biology

    Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

    doi: 10.1371/journal.pbio.3001121

    Figure Lengend Snippet: CD74 can serve as a potential target for therapy. (A, B) WT and CD74 −/− BM cells were cultured alone or incubated with blocking anti-CD74 antibody (20, 50, and 100 μg/ml). After 48 h, percent LSK from live cells was analyzed by FACS; n = 4–7, Data A and B in S8 Data . (C, D) Survival curve: 5-FU (150 mg/kg and 125mg/kg) was injected to WT and CD74 −/− mice once a week. Log-rank test *

    Article Snippet: Generation of chimeric mice For the microenvironment experiment: Lethally irradiated (950 Rad) C57BL/6 (WT) recipient mice were reconstituted with 5*106 WT or CD74−/− BM cells.

    Techniques: Cell Culture, Incubation, Blocking Assay, FACS, Injection, Mouse Assay

    Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p

    Journal: PLoS Biology

    Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

    doi: 10.1371/journal.pbio.3001121

    Figure Lengend Snippet: Accumulation of HSPCs is not CXCR4 dependent. (A, B) FACS analysis of CXCR4 expression on BM LSK and BM CD34-/LSK of WT and CD74 −/− mice, n = 7, Data A in S6 Data . Representative histograms are shown. (C–E) FACS analysis for HSPCs in the PB of WT and CD74 −/− . (C) Dot plot analysis of LSK in WT and CD74 −/− mice. (D, E) Cell number of (D) LSK and (E) CD34-LSK in 600 μl blood. WT n = 6 CD74 −/− n = 7, Data B and C in S6 Data . (F) AMD3100 (20 mg/kg −1 ) was injected to WT and CD74 −/− mice. After 2 h, percent of LSK in the PB was analyzed; n = 9–11, Data D in S6 Data . (G–I) FACS staining of WT and CD74 −/− HSPCs for Ki-67. Results are presented as: (G) percent of CD34-/LSK Ki-67 and CD34-/LSK Ki-67+ from total BM cells, Data E in S6 Data ; (H) percent of CD34+/LSK Ki-67- and CD34+/LSK Ki-67+ from total BM cells, Data F in S6 Data ; and (I) percent of Ki-67+ from CD34-/LSK and percent of Ki-67+ from CD34+LSK, n = 15, Data G in S6 Data . (J, K) Mice were fed with 0.8 mg/ml BrdU in their drinking water for 3 days, and BrdU incorporation was analyzed by FACS. Results are represented as: (J) percent of LSK BrdU- and LSK BrdU+ from total BM cells, Data H in S6 Data ; (K) percent of BrdU+ in LSK; n = 12–14, Data I in S6 Data . Bars show SEM. Unpaired two-tailed t test: * p

    Article Snippet: Generation of chimeric mice For the microenvironment experiment: Lethally irradiated (950 Rad) C57BL/6 (WT) recipient mice were reconstituted with 5*106 WT or CD74−/− BM cells.

    Techniques: FACS, Expressing, Mouse Assay, Injection, Staining, BrdU Incorporation Assay, Two Tailed Test

    CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *

    Journal: PLoS Biology

    Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

    doi: 10.1371/journal.pbio.3001121

    Figure Lengend Snippet: CD74 −/− HSPC expansion is cell intrinsic. Lethally irradiated WT or CD74 −/− mice were transplanted with either WT or CD74 −/− total BM cells. Long-term reconstitution was evaluated 16 weeks posttransplantation. Percent of total BM cells was calculated for (A) LIN -; Data A in S2 Data (B) LSK; Data B in S2 Data and (C) CD34-/LSK; Data C in S2 Data n = 5–12. Bars show SEM. Unpaired two-tailed t test *

    Article Snippet: Generation of chimeric mice For the microenvironment experiment: Lethally irradiated (950 Rad) C57BL/6 (WT) recipient mice were reconstituted with 5*106 WT or CD74−/− BM cells.

    Techniques: Irradiation, Mouse Assay, Two Tailed Test

    CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p

    Journal: PLoS Biology

    Article Title: CD74 is a regulator of hematopoietic stem cell maintenance

    doi: 10.1371/journal.pbio.3001121

    Figure Lengend Snippet: CD74 regulates the survival of HSPCs and CD18 expression. (A, B) FACS staining of WT and CD74-deficient HSPCs for ROS. (A) Results are presented as the number of ROS high cells per 10 6 cells, n = 9, Data A in S7 Data . (B) Percentage of ROS high in LSK, Data B in S7 Data . (C, D) Percent of LSK (C) Data C in S7 Data , and CD34- (D) after 6 days of NAC injections (50 mg kg −1 ); n = 5, Data D in S7 Data . (E, F) FACS analysis of HSPCs from WT and CD74 −/− mice for Annexin V (E); n = 10–12, Data E in S7 Data , and after 24 h under hypoxic (F); n = 3 (each dot represents a duplicate determination), Data F in S7 Data . (G) Ratio of Annexin V+ CD74 −/− to WT of HSPCs under hypoxic and normoxic conditions, Data G in S7 Data . (H, I) FACS analysis of HSCs from WT and CD74 −/− mice for HIF-1α; n = 7–8, Data H in S7 Data . (J) Sorted WT and CD74 −/− CD34-/LSK cells were analyzed for CD18 mRNA levels; n = 3. The bars show the DESeq2 normalized counts for the CD18 gene, Data I in S7 Data . (K) Binding of CD74–ICD to CD18 promoter and intron regions in Lin− samples. ChIP-seq analysis using anti-CD74 antibody. (L) FACS analysis of HSCs from WT and CD74 −/− mice for CD18. Graph summarizes the results of 6 mice in each group, Data J in S7 Data . (M) FACS analysis of HSCs from WT and MIF −/− mice for CD18; n = 3, Data K in S7 Data . (N) WT and CD74 −/− BM were cultured with or without the MIF inhibitor, ISO-1, for 48 h, percent CD18 on CD34-/LSK was analyzed by FACS; n = 6, Data L in S7 Data . (O) WT (CD45.1) Lin negative cells were cultured in the presence of WT (CD45.2) total BM or MIF −/− (CD45.2) total BM for 48 h. The percent CD18 on CD34-/LSK cells (CD45.1) was analyzed by FACS; n = 8, Data M in S7 Data . Bars show SEM. Unpaired two-tailed t test * p

    Article Snippet: Generation of chimeric mice For the microenvironment experiment: Lethally irradiated (950 Rad) C57BL/6 (WT) recipient mice were reconstituted with 5*106 WT or CD74−/− BM cells.

    Techniques: Expressing, FACS, Staining, Mouse Assay, Binding Assay, Chromatin Immunoprecipitation, Cell Culture, Two Tailed Test