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  • 91
    Thermo Fisher ter 119 ter119
    Cxcr4 expressing erythroid progenitors seem to migrate from the bone marrow to the spleen following a Cxcl12 gradient in immunized and nephritic mice. Spleens (A, B) and bone marrow (C, D,) of healthy (black bars, n = 3), immunized (lined bars, n = 4) and nephritic mice on day 7 (grey bars, n = 4) were evaluated for <t>Ter119</t> + Cxcr4 + cells by performing relative flow cytometry. The expression of Cxcl12 mRNA was evaluated by quantitative real-time PCR of total RNA from spleens and bone marrow of healthy, immunized and nephritic mice on day 7 (E). Data are given as means ± SEM. *p
    Ter 119 Ter119, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 91 stars, based on 47 article reviews
    Price from $9.99 to $1999.99
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    93
    Thermo Fisher ter119 pecy7
    Cxcr4 expressing erythroid progenitors seem to migrate from the bone marrow to the spleen following a Cxcl12 gradient in immunized and nephritic mice. Spleens (A, B) and bone marrow (C, D,) of healthy (black bars, n = 3), immunized (lined bars, n = 4) and nephritic mice on day 7 (grey bars, n = 4) were evaluated for <t>Ter119</t> + Cxcr4 + cells by performing relative flow cytometry. The expression of Cxcl12 mRNA was evaluated by quantitative real-time PCR of total RNA from spleens and bone marrow of healthy, immunized and nephritic mice on day 7 (E). Data are given as means ± SEM. *p
    Ter119 Pecy7, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 33 article reviews
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    90
    Thermo Fisher ter119 efluor450
    Cxcr4 expressing erythroid progenitors seem to migrate from the bone marrow to the spleen following a Cxcl12 gradient in immunized and nephritic mice. Spleens (A, B) and bone marrow (C, D,) of healthy (black bars, n = 3), immunized (lined bars, n = 4) and nephritic mice on day 7 (grey bars, n = 4) were evaluated for <t>Ter119</t> + Cxcr4 + cells by performing relative flow cytometry. The expression of Cxcl12 mRNA was evaluated by quantitative real-time PCR of total RNA from spleens and bone marrow of healthy, immunized and nephritic mice on day 7 (E). Data are given as means ± SEM. *p
    Ter119 Efluor450, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 81 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ter119 efluor450/product/Thermo Fisher
    Average 90 stars, based on 81 article reviews
    Price from $9.99 to $1999.99
    ter119 efluor450 - by Bioz Stars, 2020-05
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    88
    BioLegend ter119 ter 119
    Cxcr4 expressing erythroid progenitors seem to migrate from the bone marrow to the spleen following a Cxcl12 gradient in immunized and nephritic mice. Spleens (A, B) and bone marrow (C, D,) of healthy (black bars, n = 3), immunized (lined bars, n = 4) and nephritic mice on day 7 (grey bars, n = 4) were evaluated for <t>Ter119</t> + Cxcr4 + cells by performing relative flow cytometry. The expression of Cxcl12 mRNA was evaluated by quantitative real-time PCR of total RNA from spleens and bone marrow of healthy, immunized and nephritic mice on day 7 (E). Data are given as means ± SEM. *p
    Ter119 Ter 119, supplied by BioLegend, used in various techniques. Bioz Stars score: 88/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ter119 ter 119/product/BioLegend
    Average 88 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    ter119 ter 119 - by Bioz Stars, 2020-05
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    94
    STEMCELL Technologies Inc ter 119 ter 119 antibodies
    Cxcr4 expressing erythroid progenitors seem to migrate from the bone marrow to the spleen following a Cxcl12 gradient in immunized and nephritic mice. Spleens (A, B) and bone marrow (C, D,) of healthy (black bars, n = 3), immunized (lined bars, n = 4) and nephritic mice on day 7 (grey bars, n = 4) were evaluated for <t>Ter119</t> + Cxcr4 + cells by performing relative flow cytometry. The expression of Cxcl12 mRNA was evaluated by quantitative real-time PCR of total RNA from spleens and bone marrow of healthy, immunized and nephritic mice on day 7 (E). Data are given as means ± SEM. *p
    Ter 119 Ter 119 Antibodies, supplied by STEMCELL Technologies Inc, used in various techniques. Bioz Stars score: 94/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ter 119 ter 119 antibodies/product/STEMCELL Technologies Inc
    Average 94 stars, based on 10 article reviews
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    89
    STEMCELL Technologies Inc ter119
    Impaired Fbw7-mediated cyclin E control dysregulates mitochondrial mass during terminal erythroid cell maturation in vivo (A) Total mitochondrial mass was compared in <t>Ter119+,</t> bone marrow erythroid cells at the indicated maturation stages, based on CD71 vs. FSC. Each histogram is comprised of data obtained from two wild-type and knock-in mice. (B) Transmission electron micrograph images of peripheral blood erythrocytes (RBCs) are shown from mice of the indicated genotypes, with mitochondria retained in cyclin E T74A T393A cells indicated by bars. Peripheral RBCs with retained mitochondria were enumerated by counting approximately 1000 cells from three mice of each genotype. Mean percentage of cells with retained mitochondria and standard deviations are shown, calculated from biological replicates. (C) Bnip3L and Hbb-b1 expression in Ter119+ cells isolated at the indicated stages of erythroid maturation based on CD44/FSC gating is shown for cyclin E T74A T393A mice, compared to age- and sex-matched wild-type controls. (D) Survival of CFSE-labeled erythrocytes obtained from mice of the indicated genotypes was compared by obtaining blood samples from wild-type recipients at the indicated times, and fractions of fluorescent RBCs determined by flow cytometry. The percentage of CFSE-positive RBCs at 12 hours after injection of labeled cells was between 7%–9% of total. Data for subsequent time points are expressed as ratios to 12-hour values and represent averages from four recipients of cells from each genotype. Calculated half-lives are: 19.1 days (wild-type) and 16.5 days (cyclin E T74A T393A ), p=0.008. Inset- peripheral blood reticulocytes were enumerated in five wild-type and cyclin E T74A T393A mice, p=0.02.
    Ter119, supplied by STEMCELL Technologies Inc, used in various techniques. Bioz Stars score: 89/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ter119/product/STEMCELL Technologies Inc
    Average 89 stars, based on 19 article reviews
    Price from $9.99 to $1999.99
    ter119 - by Bioz Stars, 2020-05
    89/100 stars
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    91
    Becton Dickinson ter119 ter 119
    Impaired Fbw7-mediated cyclin E control dysregulates mitochondrial mass during terminal erythroid cell maturation in vivo (A) Total mitochondrial mass was compared in <t>Ter119+,</t> bone marrow erythroid cells at the indicated maturation stages, based on CD71 vs. FSC. Each histogram is comprised of data obtained from two wild-type and knock-in mice. (B) Transmission electron micrograph images of peripheral blood erythrocytes (RBCs) are shown from mice of the indicated genotypes, with mitochondria retained in cyclin E T74A T393A cells indicated by bars. Peripheral RBCs with retained mitochondria were enumerated by counting approximately 1000 cells from three mice of each genotype. Mean percentage of cells with retained mitochondria and standard deviations are shown, calculated from biological replicates. (C) Bnip3L and Hbb-b1 expression in Ter119+ cells isolated at the indicated stages of erythroid maturation based on CD44/FSC gating is shown for cyclin E T74A T393A mice, compared to age- and sex-matched wild-type controls. (D) Survival of CFSE-labeled erythrocytes obtained from mice of the indicated genotypes was compared by obtaining blood samples from wild-type recipients at the indicated times, and fractions of fluorescent RBCs determined by flow cytometry. The percentage of CFSE-positive RBCs at 12 hours after injection of labeled cells was between 7%–9% of total. Data for subsequent time points are expressed as ratios to 12-hour values and represent averages from four recipients of cells from each genotype. Calculated half-lives are: 19.1 days (wild-type) and 16.5 days (cyclin E T74A T393A ), p=0.008. Inset- peripheral blood reticulocytes were enumerated in five wild-type and cyclin E T74A T393A mice, p=0.02.
    Ter119 Ter 119, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 91/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ter119 ter 119/product/Becton Dickinson
    Average 91 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    ter119 ter 119 - by Bioz Stars, 2020-05
    91/100 stars
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    Image Search Results


    Cxcr4 expressing erythroid progenitors seem to migrate from the bone marrow to the spleen following a Cxcl12 gradient in immunized and nephritic mice. Spleens (A, B) and bone marrow (C, D,) of healthy (black bars, n = 3), immunized (lined bars, n = 4) and nephritic mice on day 7 (grey bars, n = 4) were evaluated for Ter119 + Cxcr4 + cells by performing relative flow cytometry. The expression of Cxcl12 mRNA was evaluated by quantitative real-time PCR of total RNA from spleens and bone marrow of healthy, immunized and nephritic mice on day 7 (E). Data are given as means ± SEM. *p

    Journal: PLoS ONE

    Article Title: The Spleen Plays No Role in Nephrotoxic Serum Nephritis, but Constitutes a Place of Compensatory Haematopoiesis

    doi: 10.1371/journal.pone.0135087

    Figure Lengend Snippet: Cxcr4 expressing erythroid progenitors seem to migrate from the bone marrow to the spleen following a Cxcl12 gradient in immunized and nephritic mice. Spleens (A, B) and bone marrow (C, D,) of healthy (black bars, n = 3), immunized (lined bars, n = 4) and nephritic mice on day 7 (grey bars, n = 4) were evaluated for Ter119 + Cxcr4 + cells by performing relative flow cytometry. The expression of Cxcl12 mRNA was evaluated by quantitative real-time PCR of total RNA from spleens and bone marrow of healthy, immunized and nephritic mice on day 7 (E). Data are given as means ± SEM. *p

    Article Snippet: For direct immunofluorescence of autologous IgG, FITC-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories) in serial dilutions and for erythroid progenitor cells FITC-conjugated antibody for Ter119+ cells (clone TER-119; eBioscience, San Diego, CA) was used and eventually mounted with mounting medium for fluorescence with DAPI (Vector Laboratories, Burlingame, CA, USA).

    Techniques: Expressing, Mouse Assay, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction

    Extramedullary haematopoiesis is observed in spleens of immunized mice and 14 days after NTS induction. HE stains and immunohistochemical stainings for CD41 and Ter119 were performed from spleens of healthy mice, immunized mice, and mice with NTS on day 14. Representative pictures are shown. Magnification x200 (HE), x400 (CD41 + ) and x600 (Ter119 + ).

    Journal: PLoS ONE

    Article Title: The Spleen Plays No Role in Nephrotoxic Serum Nephritis, but Constitutes a Place of Compensatory Haematopoiesis

    doi: 10.1371/journal.pone.0135087

    Figure Lengend Snippet: Extramedullary haematopoiesis is observed in spleens of immunized mice and 14 days after NTS induction. HE stains and immunohistochemical stainings for CD41 and Ter119 were performed from spleens of healthy mice, immunized mice, and mice with NTS on day 14. Representative pictures are shown. Magnification x200 (HE), x400 (CD41 + ) and x600 (Ter119 + ).

    Article Snippet: For direct immunofluorescence of autologous IgG, FITC-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories) in serial dilutions and for erythroid progenitor cells FITC-conjugated antibody for Ter119+ cells (clone TER-119; eBioscience, San Diego, CA) was used and eventually mounted with mounting medium for fluorescence with DAPI (Vector Laboratories, Burlingame, CA, USA).

    Techniques: Mouse Assay, Immunohistochemistry

    Sin3A has a cell-autonomous function in HSC maintenance. ( A ) Quantification of bone marrow cells from WT , MxCre + ; Sin3A +/Δ and MxCre + ; Sin3A Δ/Δ mice (n=3 mice per group) (left panel). Representative hematoxylin–eosin-stained paraffin-embedded tissue sections from bone marrow of mice with the indicated genotypes showing loss of cellularity in Sin3A-deficient bone marrow. ( B ) The bone marrow composition of the indicated genotypes (n=3 mice per group). Bone marrow cells were isolated, quantified and stained with labeled antibodies against CD11b and Gr-1 (myeloid cells), B220 and CD19 (B cells), TER119 (erythroid cells), CD41 (megakaryocytes) and CD3 (T cells) and analyzed by flow cytometry. ( C ) Flow cytometric quantification of c-kit + cells (progenitors) in the bone marrow of animals with the indicated genotypes (n=3 per group). ( D ) Flow cytometric quantification of LSK cells (lin − c-Kit + ScaI + ) in the bone marrow of animals with the indicated genotypes (n=3 per group). ( E ) The number of myeloid colonies from bone marrow of animals with the indicated genotypes cultured in methylcellulose enriched with 10 ng/mL interleukin-3, 10 ng/mL interleukin-6 and 50 ng/ml stem cell factor to induce myeloid differentiation. ( F ) Bone marrow cells from WT (Ly5.2 + ) and MxCre + ; Sin3A L/L (Ly5.1 + ) or MxCre − ; Sin3A L/L (control) mice were transplanted in a 1:1 ratio in lethally irradiated recipient mice. Bone marrow reconstitution of Ly5.1 + and Ly5.2 + populations was measured 8 weeks post-transplantation (left panel). Subsequently, mice were injected five times with pI;pC (arrows right panel). Ly5.2 + cells were monitored in the blood for 70 days (right panel). The mean value of Ly5.2 + cells was set at 100% before pI;pC injection, (n=5 mice per group). ( G ) Flow cytometric quantification of Ly5.1 + LSK in the bone marrow of the chimeras described in ( E ) (n=3 per group).

    Journal: Haematologica

    Article Title: Sin3a-associated Hdac1 and Hdac2 are essential for hematopoietic stem cell homeostasis and contribute differentially to hematopoiesis

    doi: 10.3324/haematol.2013.092643

    Figure Lengend Snippet: Sin3A has a cell-autonomous function in HSC maintenance. ( A ) Quantification of bone marrow cells from WT , MxCre + ; Sin3A +/Δ and MxCre + ; Sin3A Δ/Δ mice (n=3 mice per group) (left panel). Representative hematoxylin–eosin-stained paraffin-embedded tissue sections from bone marrow of mice with the indicated genotypes showing loss of cellularity in Sin3A-deficient bone marrow. ( B ) The bone marrow composition of the indicated genotypes (n=3 mice per group). Bone marrow cells were isolated, quantified and stained with labeled antibodies against CD11b and Gr-1 (myeloid cells), B220 and CD19 (B cells), TER119 (erythroid cells), CD41 (megakaryocytes) and CD3 (T cells) and analyzed by flow cytometry. ( C ) Flow cytometric quantification of c-kit + cells (progenitors) in the bone marrow of animals with the indicated genotypes (n=3 per group). ( D ) Flow cytometric quantification of LSK cells (lin − c-Kit + ScaI + ) in the bone marrow of animals with the indicated genotypes (n=3 per group). ( E ) The number of myeloid colonies from bone marrow of animals with the indicated genotypes cultured in methylcellulose enriched with 10 ng/mL interleukin-3, 10 ng/mL interleukin-6 and 50 ng/ml stem cell factor to induce myeloid differentiation. ( F ) Bone marrow cells from WT (Ly5.2 + ) and MxCre + ; Sin3A L/L (Ly5.1 + ) or MxCre − ; Sin3A L/L (control) mice were transplanted in a 1:1 ratio in lethally irradiated recipient mice. Bone marrow reconstitution of Ly5.1 + and Ly5.2 + populations was measured 8 weeks post-transplantation (left panel). Subsequently, mice were injected five times with pI;pC (arrows right panel). Ly5.2 + cells were monitored in the blood for 70 days (right panel). The mean value of Ly5.2 + cells was set at 100% before pI;pC injection, (n=5 mice per group). ( G ) Flow cytometric quantification of Ly5.1 + LSK in the bone marrow of the chimeras described in ( E ) (n=3 per group).

    Article Snippet: LSK cells and progenitors were stained as described previously with the following labeled antibodies: IL7Rα-biotin, CD3-biotin, B220-biotin, CD11b-biotin, TER119-biotin and Ly6G-biotin (eBioscience) followed by streptavidin-PerCP-Cy5.5, CD34-FITC FcgRII/III-PE (eBioscience), ScaI-PacificBlue (Biolegend), CD34-FITC, IL7Ra-PE, and c-kit-APC (BD Biosciences).

    Techniques: Mouse Assay, Staining, Isolation, Labeling, Flow Cytometry, Cytometry, Cell Culture, Irradiation, Transplantation Assay, Injection

    Hdac1 and Hdac2 do not compensate for each other equally in hematopoiesis. ( A ) Western blot analysis for Hdac1 and Hdac2 protein levels in the bone marrow of the mice with the indicated genotypes. Pictures are representative images of the results from three or more independent mice. Tubulin was used a loading control. ( B ) Kaplan-Meier survival curves demonstrating the survival of WT , MxCre + ; Hdac1 +/Δ Hdac2 Δ/Δ and MxCre + ; Hdac1 Δ/Δ Hdac2 +/Δ mice after five injections of pI;pC (at least 3 mice per group). ( C ) Quantification of erythrolysed bone marrow of indicated genotypes (n=3 mice per group). ( D ) Number of bone marrow cells after erythrocyte lysis of WT , MxCre + ; Hdac1 +/Δ Hdac2 Δ/Δ and MxCre + ;Hdac1 Δ/Δ Hdac2 +/Δ mice (n=3 mice per group). Bone marrow cells were isolated, quantified and stained with labeled antibodies against CD11b and Gr-1 (myeloid cells), B220 and CD19 (B cells), TER119 (erythroid cells), CD41 (megakaryocytes) and CD3 (T cells) and analyzed by flow cytometry. ( E ) The number of pre-B-cell and myeloid colonies from bone marrow of WT , MxCre + ; Hdac1 Δ/ Δ Hdac2 +/Δ and MxCre + ; Hdac1 +/Δ Hdac2 Δ/Δ mice cultured in methylcellulose enriched with either 10 ng/mL interleukin-3, 10 ng/mL interleukin-6 and 50 ng/mL stem cell factor to induce myeloid differentiation or with 10 ng/mL interleukin-7 to induce differentiation into pre-B cells.

    Journal: Haematologica

    Article Title: Sin3a-associated Hdac1 and Hdac2 are essential for hematopoietic stem cell homeostasis and contribute differentially to hematopoiesis

    doi: 10.3324/haematol.2013.092643

    Figure Lengend Snippet: Hdac1 and Hdac2 do not compensate for each other equally in hematopoiesis. ( A ) Western blot analysis for Hdac1 and Hdac2 protein levels in the bone marrow of the mice with the indicated genotypes. Pictures are representative images of the results from three or more independent mice. Tubulin was used a loading control. ( B ) Kaplan-Meier survival curves demonstrating the survival of WT , MxCre + ; Hdac1 +/Δ Hdac2 Δ/Δ and MxCre + ; Hdac1 Δ/Δ Hdac2 +/Δ mice after five injections of pI;pC (at least 3 mice per group). ( C ) Quantification of erythrolysed bone marrow of indicated genotypes (n=3 mice per group). ( D ) Number of bone marrow cells after erythrocyte lysis of WT , MxCre + ; Hdac1 +/Δ Hdac2 Δ/Δ and MxCre + ;Hdac1 Δ/Δ Hdac2 +/Δ mice (n=3 mice per group). Bone marrow cells were isolated, quantified and stained with labeled antibodies against CD11b and Gr-1 (myeloid cells), B220 and CD19 (B cells), TER119 (erythroid cells), CD41 (megakaryocytes) and CD3 (T cells) and analyzed by flow cytometry. ( E ) The number of pre-B-cell and myeloid colonies from bone marrow of WT , MxCre + ; Hdac1 Δ/ Δ Hdac2 +/Δ and MxCre + ; Hdac1 +/Δ Hdac2 Δ/Δ mice cultured in methylcellulose enriched with either 10 ng/mL interleukin-3, 10 ng/mL interleukin-6 and 50 ng/mL stem cell factor to induce myeloid differentiation or with 10 ng/mL interleukin-7 to induce differentiation into pre-B cells.

    Article Snippet: LSK cells and progenitors were stained as described previously with the following labeled antibodies: IL7Rα-biotin, CD3-biotin, B220-biotin, CD11b-biotin, TER119-biotin and Ly6G-biotin (eBioscience) followed by streptavidin-PerCP-Cy5.5, CD34-FITC FcgRII/III-PE (eBioscience), ScaI-PacificBlue (Biolegend), CD34-FITC, IL7Ra-PE, and c-kit-APC (BD Biosciences).

    Techniques: Western Blot, Mouse Assay, Lysis, Isolation, Staining, Labeling, Flow Cytometry, Cytometry, Cell Culture

    Hdac1 and Hdac2 are collectively essential for hematopoiesis. ( A ) Splenocyte and thymocyte counts of the indicated groups (n=3 per group). ( B ) Quantification of bone marrow cells after erythrocyte lysis. ( C ) The bone marrow composition of WT , MxCre + ; Hdac1 Δ/Δ , MxCre + ; Hdac2 Δ/Δ and DKO mice 8 days after the last pl:pC injection (n=3 per group). Bone marrow cells were isolated, quantified and stained with labeled antibodies against CD11b and Gr-1 (myeloid cells), B220 and CD19 (B cells), TER119 (erythroid cells), CD41 (megakaryocytes) and CD3 (T cells) and analyzed by flow cytometry. ( D ) Apoptosis in the bone marrow of control and DKO mice was analyzed by flow cytometry using a labeled antibody against caspase-3. Histograms are representative of three experiments per group; the average and standard deviation are presented on top. ( E ) Flow cytometric quantification of c-Kit + cells (progenitors) and LSK (Lin − ;c-Kit + ;ScaI + ) cells in the bone marrow of WT and DKO mice (n=3 per group). ( F ) The number of myeloid and pre-B-cell colonies from bone marrow of WT and DKO mice cultured in methylcellulose enriched with either 10 ng/mL interleukin–3, 10 ng/mL interleukin-6 and 50 ng/mL stem cell factor to induce myeloid differentiation or with 10 ng/mL interleukin-7 to induce differentiation into pre-B cells.

    Journal: Haematologica

    Article Title: Sin3a-associated Hdac1 and Hdac2 are essential for hematopoietic stem cell homeostasis and contribute differentially to hematopoiesis

    doi: 10.3324/haematol.2013.092643

    Figure Lengend Snippet: Hdac1 and Hdac2 are collectively essential for hematopoiesis. ( A ) Splenocyte and thymocyte counts of the indicated groups (n=3 per group). ( B ) Quantification of bone marrow cells after erythrocyte lysis. ( C ) The bone marrow composition of WT , MxCre + ; Hdac1 Δ/Δ , MxCre + ; Hdac2 Δ/Δ and DKO mice 8 days after the last pl:pC injection (n=3 per group). Bone marrow cells were isolated, quantified and stained with labeled antibodies against CD11b and Gr-1 (myeloid cells), B220 and CD19 (B cells), TER119 (erythroid cells), CD41 (megakaryocytes) and CD3 (T cells) and analyzed by flow cytometry. ( D ) Apoptosis in the bone marrow of control and DKO mice was analyzed by flow cytometry using a labeled antibody against caspase-3. Histograms are representative of three experiments per group; the average and standard deviation are presented on top. ( E ) Flow cytometric quantification of c-Kit + cells (progenitors) and LSK (Lin − ;c-Kit + ;ScaI + ) cells in the bone marrow of WT and DKO mice (n=3 per group). ( F ) The number of myeloid and pre-B-cell colonies from bone marrow of WT and DKO mice cultured in methylcellulose enriched with either 10 ng/mL interleukin–3, 10 ng/mL interleukin-6 and 50 ng/mL stem cell factor to induce myeloid differentiation or with 10 ng/mL interleukin-7 to induce differentiation into pre-B cells.

    Article Snippet: LSK cells and progenitors were stained as described previously with the following labeled antibodies: IL7Rα-biotin, CD3-biotin, B220-biotin, CD11b-biotin, TER119-biotin and Ly6G-biotin (eBioscience) followed by streptavidin-PerCP-Cy5.5, CD34-FITC FcgRII/III-PE (eBioscience), ScaI-PacificBlue (Biolegend), CD34-FITC, IL7Ra-PE, and c-kit-APC (BD Biosciences).

    Techniques: Lysis, Mouse Assay, Injection, Isolation, Staining, Labeling, Flow Cytometry, Cytometry, Standard Deviation, Cell Culture

    Erythropoiesis is severely affected only in MxCre + ; Hdac1 Δ/Δ Hdac2 +/Δ mice. ( A ) Three WT and three enlarged MxCre + ; Hdac1 Δ/Δ Hdac2 +/Δ spleens. ( B ) Splenocyte numbers in WT and MxCre + ; Hdac1 Δ/Δ Hdac2 +/Δ mice (n=3 mice per group). ( C ) After erythrocyte lysis, which only affects enucleated cells, the number of nucleated erythrocytes in spleens from WT and MxCre + ; Hdac1 Δ/Δ Hdac2 +/Δ mice was determined (n=3 mice per group). ( D ) The number of myeloid colonies from spleen of WT and MxCre + ; Hdac1 Δ/Δ Hdac2 +/Δ mice cultured in methylcellulose enriched with 10 ng/mL interleukin-3, 10 ng/mL interleukin-6 and 50 ng/mL stem cell factor to induce myeloid differentiation. ( E ) Hematoxylin-eosinstained histological liver sections of pI;pC-treated mice with indicated genotypes. Only MxCre + ; Hdac1 Δ/Δ Hdac2 +/Δ livers showed the presence of nucleated erythroid cells ( F ) Erythroid development in the bone marrow of WT , MxCre + ; Hdac1 Δ/Δ Hdac2 +/Δ mice was analyzed by flow cytometry using the labeled erythroid markers CD71 and Ter119. I, II, III and IV represent pro-erythroblasts, basophilic, polychromatic and orthochromatic erythroblasts, respectively (left panel). Absolute numbers are plotted (right panel).

    Journal: Haematologica

    Article Title: Sin3a-associated Hdac1 and Hdac2 are essential for hematopoietic stem cell homeostasis and contribute differentially to hematopoiesis

    doi: 10.3324/haematol.2013.092643

    Figure Lengend Snippet: Erythropoiesis is severely affected only in MxCre + ; Hdac1 Δ/Δ Hdac2 +/Δ mice. ( A ) Three WT and three enlarged MxCre + ; Hdac1 Δ/Δ Hdac2 +/Δ spleens. ( B ) Splenocyte numbers in WT and MxCre + ; Hdac1 Δ/Δ Hdac2 +/Δ mice (n=3 mice per group). ( C ) After erythrocyte lysis, which only affects enucleated cells, the number of nucleated erythrocytes in spleens from WT and MxCre + ; Hdac1 Δ/Δ Hdac2 +/Δ mice was determined (n=3 mice per group). ( D ) The number of myeloid colonies from spleen of WT and MxCre + ; Hdac1 Δ/Δ Hdac2 +/Δ mice cultured in methylcellulose enriched with 10 ng/mL interleukin-3, 10 ng/mL interleukin-6 and 50 ng/mL stem cell factor to induce myeloid differentiation. ( E ) Hematoxylin-eosinstained histological liver sections of pI;pC-treated mice with indicated genotypes. Only MxCre + ; Hdac1 Δ/Δ Hdac2 +/Δ livers showed the presence of nucleated erythroid cells ( F ) Erythroid development in the bone marrow of WT , MxCre + ; Hdac1 Δ/Δ Hdac2 +/Δ mice was analyzed by flow cytometry using the labeled erythroid markers CD71 and Ter119. I, II, III and IV represent pro-erythroblasts, basophilic, polychromatic and orthochromatic erythroblasts, respectively (left panel). Absolute numbers are plotted (right panel).

    Article Snippet: LSK cells and progenitors were stained as described previously with the following labeled antibodies: IL7Rα-biotin, CD3-biotin, B220-biotin, CD11b-biotin, TER119-biotin and Ly6G-biotin (eBioscience) followed by streptavidin-PerCP-Cy5.5, CD34-FITC FcgRII/III-PE (eBioscience), ScaI-PacificBlue (Biolegend), CD34-FITC, IL7Ra-PE, and c-kit-APC (BD Biosciences).

    Techniques: Mouse Assay, Lysis, Cell Culture, Flow Cytometry, Cytometry, Labeling

    Infection-induced persistent GCs generate highly mutated B cells. (A) HA-binding GC B cells [Live/Dead-Dump − (IgM − IgD − Gr-1 − CD3 − CD5 − CD11b − CD11c − CD43 − CD90 − CD93 − TER-119 − F4/80 − CD117 − CD138 − Live/Dead-Aqua − )CD38 dull B220 + ] were gated, and the intracellular expression of Bcl-6 protein was evaluated by flow cytometry. The data are representative of three independent experiments. (B) The numbers of HA-binding GC B cells (PI-Dump − CD38 dull B220 + ) in each organ are plotted at the indicated time points (mean ± SD; n = 4–9). Over 20,000,000 events were analyzed from individual spleens, and the cells within enlarged B220 versus dump gate were collected for further analysis at days 40 and 60 as shown in Fig. S1 . The data are representative of two independent experiments. (C) Frozen sections (8 µm) were prepared from mice ( n = 7) at day 30 after infection and stained with B220/CD4/GL7. In total, 569 (lungs), 272 (MLNs), and 1,439 (spleens) GL7 + B220 + GC B cell clusters were scanned, and representative images on mean size are presented. Bars, 100 µm. (D) RNAs were extracted from the indicated organs ( n = 6), serially diluted at threefold dilution, and subjected to one-step RT-PCR analysis for NP (40 cycles) and β-actin (23 cycles) genes. The data are representative of two independent experiments. (E) After intraperitoneal EdU injection (1 mg) at day 30 after infection, the EdU uptake of HA-binding GL7 + cells among Live/Dead-Dump − B220 + gate (as shown in A) in the indicated organs was analyzed 8 h later. Representative flow data for EdU labeling are presented, and the EdU + ratios of HA-binding GC B cells are plotted. Each circle represents the result from an individual mouse. The data are representative of three independent experiments. ***, P

    Journal: The Journal of Experimental Medicine

    Article Title: Distinct germinal center selection at local sites shapes memory B cell response to viral escape

    doi: 10.1084/jem.20142284

    Figure Lengend Snippet: Infection-induced persistent GCs generate highly mutated B cells. (A) HA-binding GC B cells [Live/Dead-Dump − (IgM − IgD − Gr-1 − CD3 − CD5 − CD11b − CD11c − CD43 − CD90 − CD93 − TER-119 − F4/80 − CD117 − CD138 − Live/Dead-Aqua − )CD38 dull B220 + ] were gated, and the intracellular expression of Bcl-6 protein was evaluated by flow cytometry. The data are representative of three independent experiments. (B) The numbers of HA-binding GC B cells (PI-Dump − CD38 dull B220 + ) in each organ are plotted at the indicated time points (mean ± SD; n = 4–9). Over 20,000,000 events were analyzed from individual spleens, and the cells within enlarged B220 versus dump gate were collected for further analysis at days 40 and 60 as shown in Fig. S1 . The data are representative of two independent experiments. (C) Frozen sections (8 µm) were prepared from mice ( n = 7) at day 30 after infection and stained with B220/CD4/GL7. In total, 569 (lungs), 272 (MLNs), and 1,439 (spleens) GL7 + B220 + GC B cell clusters were scanned, and representative images on mean size are presented. Bars, 100 µm. (D) RNAs were extracted from the indicated organs ( n = 6), serially diluted at threefold dilution, and subjected to one-step RT-PCR analysis for NP (40 cycles) and β-actin (23 cycles) genes. The data are representative of two independent experiments. (E) After intraperitoneal EdU injection (1 mg) at day 30 after infection, the EdU uptake of HA-binding GL7 + cells among Live/Dead-Dump − B220 + gate (as shown in A) in the indicated organs was analyzed 8 h later. Representative flow data for EdU labeling are presented, and the EdU + ratios of HA-binding GC B cells are plotted. Each circle represents the result from an individual mouse. The data are representative of three independent experiments. ***, P

    Article Snippet: Anti-IgM (II/41)–biotin, anti-IgD (11-26c)–biotin, anti-B220/CD45R (RA3-6B2)–biotin, anti-CD93 (AA4.1)–biotin, anti-Thy1.2/CD90.2 (53-2.1)–biotin, anti-CD3 (145-2C11)–biotin, anti–Gr-1 (RB6-8C5)–biotin, anti-F4/80 (BM8)–biotin, anti-CD11b (M1/70)–biotin, anti-CD11c (N418)–biotin, anti-CD117 (2B8)–biotin, anti-CD8α (53-6.7)–biotin, anti–TER-119 (TER-119)–biotin, anti-CD5 (53-7.3)–biotin, anti-CD4 (GK1.5)–biotin, anti-IgD (11-26)–PE, and anti-CD4 (RM4-5)–APC were purchased from eBioscience.

    Techniques: Infection, Binding Assay, Expressing, Flow Cytometry, Cytometry, Mouse Assay, Staining, Reverse Transcription Polymerase Chain Reaction, Injection, Labeling

    Simultaneous staining by two HA probes segregates cross-reactive and strain-specific memory B cells. (A) The numbers of X31/Uruguay HA-binding CD38 + (memory) and CD38 dull (GC) B cells among the PI-Dump − (IgM − IgD − Gr-1 − CD3 − CD5 − CD11b − CD11c − CD43 − CD90 − CD93 − TER-119 − F4/80 − CD117 − CD138 − PI − )B220 + fraction were counted from naive (black) and X31-infected (red) mice and plotted per organ. (B) Virus- and HA-binding memory phenotype B cells were enumerated in naive spleens with or without neuraminidase (NA) treatment. **, P

    Journal: The Journal of Experimental Medicine

    Article Title: Distinct germinal center selection at local sites shapes memory B cell response to viral escape

    doi: 10.1084/jem.20142284

    Figure Lengend Snippet: Simultaneous staining by two HA probes segregates cross-reactive and strain-specific memory B cells. (A) The numbers of X31/Uruguay HA-binding CD38 + (memory) and CD38 dull (GC) B cells among the PI-Dump − (IgM − IgD − Gr-1 − CD3 − CD5 − CD11b − CD11c − CD43 − CD90 − CD93 − TER-119 − F4/80 − CD117 − CD138 − PI − )B220 + fraction were counted from naive (black) and X31-infected (red) mice and plotted per organ. (B) Virus- and HA-binding memory phenotype B cells were enumerated in naive spleens with or without neuraminidase (NA) treatment. **, P

    Article Snippet: Anti-IgM (II/41)–biotin, anti-IgD (11-26c)–biotin, anti-B220/CD45R (RA3-6B2)–biotin, anti-CD93 (AA4.1)–biotin, anti-Thy1.2/CD90.2 (53-2.1)–biotin, anti-CD3 (145-2C11)–biotin, anti–Gr-1 (RB6-8C5)–biotin, anti-F4/80 (BM8)–biotin, anti-CD11b (M1/70)–biotin, anti-CD11c (N418)–biotin, anti-CD117 (2B8)–biotin, anti-CD8α (53-6.7)–biotin, anti–TER-119 (TER-119)–biotin, anti-CD5 (53-7.3)–biotin, anti-CD4 (GK1.5)–biotin, anti-IgD (11-26)–PE, and anti-CD4 (RM4-5)–APC were purchased from eBioscience.

    Techniques: Staining, Binding Assay, Infection, Mouse Assay

    Impaired Fbw7-mediated cyclin E control dysregulates mitochondrial mass during terminal erythroid cell maturation in vivo (A) Total mitochondrial mass was compared in Ter119+, bone marrow erythroid cells at the indicated maturation stages, based on CD71 vs. FSC. Each histogram is comprised of data obtained from two wild-type and knock-in mice. (B) Transmission electron micrograph images of peripheral blood erythrocytes (RBCs) are shown from mice of the indicated genotypes, with mitochondria retained in cyclin E T74A T393A cells indicated by bars. Peripheral RBCs with retained mitochondria were enumerated by counting approximately 1000 cells from three mice of each genotype. Mean percentage of cells with retained mitochondria and standard deviations are shown, calculated from biological replicates. (C) Bnip3L and Hbb-b1 expression in Ter119+ cells isolated at the indicated stages of erythroid maturation based on CD44/FSC gating is shown for cyclin E T74A T393A mice, compared to age- and sex-matched wild-type controls. (D) Survival of CFSE-labeled erythrocytes obtained from mice of the indicated genotypes was compared by obtaining blood samples from wild-type recipients at the indicated times, and fractions of fluorescent RBCs determined by flow cytometry. The percentage of CFSE-positive RBCs at 12 hours after injection of labeled cells was between 7%–9% of total. Data for subsequent time points are expressed as ratios to 12-hour values and represent averages from four recipients of cells from each genotype. Calculated half-lives are: 19.1 days (wild-type) and 16.5 days (cyclin E T74A T393A ), p=0.008. Inset- peripheral blood reticulocytes were enumerated in five wild-type and cyclin E T74A T393A mice, p=0.02.

    Journal: Oncogene

    Article Title: Fbw7-dependent cyclin E regulation ensures terminal maturation of bone marrow erythroid cells by restraining oxidative metabolism

    doi: 10.1038/onc.2013.289

    Figure Lengend Snippet: Impaired Fbw7-mediated cyclin E control dysregulates mitochondrial mass during terminal erythroid cell maturation in vivo (A) Total mitochondrial mass was compared in Ter119+, bone marrow erythroid cells at the indicated maturation stages, based on CD71 vs. FSC. Each histogram is comprised of data obtained from two wild-type and knock-in mice. (B) Transmission electron micrograph images of peripheral blood erythrocytes (RBCs) are shown from mice of the indicated genotypes, with mitochondria retained in cyclin E T74A T393A cells indicated by bars. Peripheral RBCs with retained mitochondria were enumerated by counting approximately 1000 cells from three mice of each genotype. Mean percentage of cells with retained mitochondria and standard deviations are shown, calculated from biological replicates. (C) Bnip3L and Hbb-b1 expression in Ter119+ cells isolated at the indicated stages of erythroid maturation based on CD44/FSC gating is shown for cyclin E T74A T393A mice, compared to age- and sex-matched wild-type controls. (D) Survival of CFSE-labeled erythrocytes obtained from mice of the indicated genotypes was compared by obtaining blood samples from wild-type recipients at the indicated times, and fractions of fluorescent RBCs determined by flow cytometry. The percentage of CFSE-positive RBCs at 12 hours after injection of labeled cells was between 7%–9% of total. Data for subsequent time points are expressed as ratios to 12-hour values and represent averages from four recipients of cells from each genotype. Calculated half-lives are: 19.1 days (wild-type) and 16.5 days (cyclin E T74A T393A ), p=0.008. Inset- peripheral blood reticulocytes were enumerated in five wild-type and cyclin E T74A T393A mice, p=0.02.

    Article Snippet: For separation of Ter119+ cells from whole bone marrow, cells were incubated with PE-Ter119 antibody then immunomagnetic beads (PE Selection Kit, Stem Cell Technology).

    Techniques: In Vivo, Knock-In, Mouse Assay, Transmission Assay, Expressing, Isolation, Labeling, Flow Cytometry, Cytometry, Injection

    Increased reactive oxygen species, induced by cyclin E or exogenously administered, induce histone H3 lysine 9 hyper-methylation in differentiated erythroid cells and disrupt GATA-1 interactions with target genes (A) Chromatin immunoprecipitation (ChIP) analysis of GATA-1 occupancy at the Hbb-b1 promoter and Bnip3L (first intron) and a 10kb-downstream region was performed in G1ER cells transduced with the indicated constructs and induced for differentiation for 24 hours. Enrichment, relative to IgG control, is shown for one representative of three independent sets of transductions and ChIPs. Corresponding enrichment for tri-methylated H3K9 (H3K9me3) at each gene is displayed below GATA-1 occupancy data . (B) Expression of Hbb-b1 and Bnip3L was measured in differentiated G1ER cells, following treatment with hydrogen peroxide (H 2 O 2 ) at the indicated doses. Exogenous peroxide was added eight hours after induction of differentiation with beta-estradiol, and cells were collected at 48 hours. Non-viable cells were excluded from collection by detecting retained propidium iodide. Inset shows cell pellets with H 2 O 2 administration at the indicated doses (in μM). (C) Lysates were prepared from differentiated G1ER cells, following treatment with hydrogen peroxide (H 2 O 2 ) for immunoblot assays. Quantitation of each histone modification was performed with normalization to total H3 signal. Shown is a representative result from three independent experiments. Mean increases in Mitosox signal relative to untreated cells were obtained using FlowJo and are expressed relative to untreated control. (D) Ter119+ bone marrow cell lysates from mice of the indicated genotypes were electrophoresed and immunoblotted as shown. Total H3 is shown as loading control. Displayed data are representative of three independent experiments. (E) G1ER cells, transduced as shown, were collected at the indicated time points during erythroid differentiation then lysed for immunoblot analyses. (en – endogenous cyclin E signal, * - retrovirally expressed cyclin E). Mitochondrial superoxide levels during differentiation were measured as for Figure 4D and mean Mitosox signal calculated for cyclin E-AA-transduced cells is expressed relative to the corresponding MIGR1-transduced cell time point. (F) Endogenous H3K9 demethylation activity was measured in lysates prepared from G1ER cells transduced or treated as indicated after differentiation in culture for the indicated times, using a quantitative fluorometric assay. Cyclin E(AA)-transduced cell lysates yielded readings above substrate-free baseline readings, though activity calculated using formula described in Methods obtains negative value. Results displayed are from a representative assay of two independent experiments.

    Journal: Oncogene

    Article Title: Fbw7-dependent cyclin E regulation ensures terminal maturation of bone marrow erythroid cells by restraining oxidative metabolism

    doi: 10.1038/onc.2013.289

    Figure Lengend Snippet: Increased reactive oxygen species, induced by cyclin E or exogenously administered, induce histone H3 lysine 9 hyper-methylation in differentiated erythroid cells and disrupt GATA-1 interactions with target genes (A) Chromatin immunoprecipitation (ChIP) analysis of GATA-1 occupancy at the Hbb-b1 promoter and Bnip3L (first intron) and a 10kb-downstream region was performed in G1ER cells transduced with the indicated constructs and induced for differentiation for 24 hours. Enrichment, relative to IgG control, is shown for one representative of three independent sets of transductions and ChIPs. Corresponding enrichment for tri-methylated H3K9 (H3K9me3) at each gene is displayed below GATA-1 occupancy data . (B) Expression of Hbb-b1 and Bnip3L was measured in differentiated G1ER cells, following treatment with hydrogen peroxide (H 2 O 2 ) at the indicated doses. Exogenous peroxide was added eight hours after induction of differentiation with beta-estradiol, and cells were collected at 48 hours. Non-viable cells were excluded from collection by detecting retained propidium iodide. Inset shows cell pellets with H 2 O 2 administration at the indicated doses (in μM). (C) Lysates were prepared from differentiated G1ER cells, following treatment with hydrogen peroxide (H 2 O 2 ) for immunoblot assays. Quantitation of each histone modification was performed with normalization to total H3 signal. Shown is a representative result from three independent experiments. Mean increases in Mitosox signal relative to untreated cells were obtained using FlowJo and are expressed relative to untreated control. (D) Ter119+ bone marrow cell lysates from mice of the indicated genotypes were electrophoresed and immunoblotted as shown. Total H3 is shown as loading control. Displayed data are representative of three independent experiments. (E) G1ER cells, transduced as shown, were collected at the indicated time points during erythroid differentiation then lysed for immunoblot analyses. (en – endogenous cyclin E signal, * - retrovirally expressed cyclin E). Mitochondrial superoxide levels during differentiation were measured as for Figure 4D and mean Mitosox signal calculated for cyclin E-AA-transduced cells is expressed relative to the corresponding MIGR1-transduced cell time point. (F) Endogenous H3K9 demethylation activity was measured in lysates prepared from G1ER cells transduced or treated as indicated after differentiation in culture for the indicated times, using a quantitative fluorometric assay. Cyclin E(AA)-transduced cell lysates yielded readings above substrate-free baseline readings, though activity calculated using formula described in Methods obtains negative value. Results displayed are from a representative assay of two independent experiments.

    Article Snippet: For separation of Ter119+ cells from whole bone marrow, cells were incubated with PE-Ter119 antibody then immunomagnetic beads (PE Selection Kit, Stem Cell Technology).

    Techniques: Methylation, Chromatin Immunoprecipitation, Transduction, Construct, Expressing, Quantitation Assay, Modification, Mouse Assay, Activity Assay

    Cell cycle arrest and survival during terminal erythroid maturation requires Fbw7-dependent cyclin E regulation (A, B) Cell cycle distributions (A) and apoptotic populations using Annexin V staining (B) were measured in bone marrow erythroid cells gated based on CD44/Ter119/FSC as in Figure 1B . Averaged data from six wild-type and cyclin E knock-in mice are displayed. Error bars indicate standard deviation. ** - p-value

    Journal: Oncogene

    Article Title: Fbw7-dependent cyclin E regulation ensures terminal maturation of bone marrow erythroid cells by restraining oxidative metabolism

    doi: 10.1038/onc.2013.289

    Figure Lengend Snippet: Cell cycle arrest and survival during terminal erythroid maturation requires Fbw7-dependent cyclin E regulation (A, B) Cell cycle distributions (A) and apoptotic populations using Annexin V staining (B) were measured in bone marrow erythroid cells gated based on CD44/Ter119/FSC as in Figure 1B . Averaged data from six wild-type and cyclin E knock-in mice are displayed. Error bars indicate standard deviation. ** - p-value

    Article Snippet: For separation of Ter119+ cells from whole bone marrow, cells were incubated with PE-Ter119 antibody then immunomagnetic beads (PE Selection Kit, Stem Cell Technology).

    Techniques: Staining, Knock-In, Mouse Assay, Standard Deviation

    Dysregulated cyclin E in bone marrow erythroid progenitor cells activates p53 and the DNA damage response pathway (A) Immunoblot analyses for cyclin E and components of the p53-dependent DNA damage response were performed using primary bone marrow cell lysates prepared after immunomagnetic separation based on Ter119 expression. HSC70 is shown as loading control; phospho-Chk1 detection was performed using same lysates as rest of immunoblot, but these were electrophoresed separately. (B) DNA damage foci were enumerated in primary Ter119+ bone marrow cells of the indicated genotypes using serine 139-phosphorylated H2AX (γH2AX) detection by flow cytometry. Shown is a representative comparison from two independent experiments. (C) Expression of the indicated p53 gene targets in Ter119+ CD71-high erythroid cells was measured by RT-PCR. Each colored bar represents averaged value of triplicate RT-PCR assays from a single knock-in mouse, expressed relative to expression in cells from age- and sex-matched wild-type controls. (D) Erythroid maturation was studied in ten wild-type recipient mice transplanted with 1×10 6 cells of the indicated genotypes and 5×10 5 wild-type (CD45.1) bone marrow cells. Five weeks following engraftment (equivalent for wild-type and knock-in donor cells), half the recipient mice were treated with pIpC, and sixteen weeks later, recipients were euthanized for study. Ter119 vs. CD71 immunophenotyping profiles from bone marrows are shown for two representative sets of mice along with PCR genotyping (far right) of bone marrow cells demonstrating efficient excision of floxed p53 allele following pIpC injections (* - non-specific amplicon generated by PCR). The p-values are calculated from paired t test comparing ratios of low CD71-, Ter119+ and high CD71-, Ter119+ erythroid progenitors. (E) Mean peripheral red blood cell counts and hemoglobin concentrations are shown for recipients of purified hematopoietic stem cells isolated from donor mice, three months following transplantation (n=24). Engraftment measured by peripheral blood CD45.2-positive cell enumeration was found to be comparable across all donor HSC groups (70–87%).

    Journal: Oncogene

    Article Title: Fbw7-dependent cyclin E regulation ensures terminal maturation of bone marrow erythroid cells by restraining oxidative metabolism

    doi: 10.1038/onc.2013.289

    Figure Lengend Snippet: Dysregulated cyclin E in bone marrow erythroid progenitor cells activates p53 and the DNA damage response pathway (A) Immunoblot analyses for cyclin E and components of the p53-dependent DNA damage response were performed using primary bone marrow cell lysates prepared after immunomagnetic separation based on Ter119 expression. HSC70 is shown as loading control; phospho-Chk1 detection was performed using same lysates as rest of immunoblot, but these were electrophoresed separately. (B) DNA damage foci were enumerated in primary Ter119+ bone marrow cells of the indicated genotypes using serine 139-phosphorylated H2AX (γH2AX) detection by flow cytometry. Shown is a representative comparison from two independent experiments. (C) Expression of the indicated p53 gene targets in Ter119+ CD71-high erythroid cells was measured by RT-PCR. Each colored bar represents averaged value of triplicate RT-PCR assays from a single knock-in mouse, expressed relative to expression in cells from age- and sex-matched wild-type controls. (D) Erythroid maturation was studied in ten wild-type recipient mice transplanted with 1×10 6 cells of the indicated genotypes and 5×10 5 wild-type (CD45.1) bone marrow cells. Five weeks following engraftment (equivalent for wild-type and knock-in donor cells), half the recipient mice were treated with pIpC, and sixteen weeks later, recipients were euthanized for study. Ter119 vs. CD71 immunophenotyping profiles from bone marrows are shown for two representative sets of mice along with PCR genotyping (far right) of bone marrow cells demonstrating efficient excision of floxed p53 allele following pIpC injections (* - non-specific amplicon generated by PCR). The p-values are calculated from paired t test comparing ratios of low CD71-, Ter119+ and high CD71-, Ter119+ erythroid progenitors. (E) Mean peripheral red blood cell counts and hemoglobin concentrations are shown for recipients of purified hematopoietic stem cells isolated from donor mice, three months following transplantation (n=24). Engraftment measured by peripheral blood CD45.2-positive cell enumeration was found to be comparable across all donor HSC groups (70–87%).

    Article Snippet: For separation of Ter119+ cells from whole bone marrow, cells were incubated with PE-Ter119 antibody then immunomagnetic beads (PE Selection Kit, Stem Cell Technology).

    Techniques: Immunomagnetic Separation, Expressing, Flow Cytometry, Cytometry, Reverse Transcription Polymerase Chain Reaction, Knock-In, Mouse Assay, Polymerase Chain Reaction, Amplification, Generated, Purification, Isolation, Transplantation Assay

    Oxidative metabolism and mitochondrial ROS are increased in cyclin E T74A T393A erythroid cells (A) Oxidative consumption rates coupled to mitochondrial ATP production were assayed in primary Ter119+ cells from three sets of mice of the indicated genotypes using a Seahorse Extracellular Flux Analyzer. (wt= wild-type, T74A T393A= cyclin E T74A T393A cells; ** - 4×10 5 , * - 3×10 5 cells analyzed in independent assays). Error bars indicate standard error with four replicates per sample. (B) Expression of the indicated genes in purified Ter119+ CD71-high erythroid cells was measured by RT-PCR. Each grey bar represents averaged value of triplicate assays from a single knock-in mouse, expressed relative to expression in cells from age- and sex-matched wild-type controls. Inset shows total and phospho-Rb expression from similarly isolated cells primary erythroid cells. (C) Left column - bone marrow Ter119+ cells were subdivided into distinct morphologic subpopulations (II–V) based on CD71 expression vs. forward scatter (FSC) 14 , with relative abundances indicated. Right columns - intracellular reactive oxygen species were measured in the indicated erythroid cell subsets using cell-permeable 2′,7′-dichlorodihydrofluorescein diacetate (H 2 DCFDA), enabling detection by flow cytometry. Comparisons between mean fluorescence signals for bone marrow erythroid cells of four mice from each of the indicated genotype groups were made and p-values calculated using paired t-tests. (D) Left – Representative comparison is shown of mitochondrial superoxide levels measured in bone marrow erythroid cells from three pairs of mice at the indicated stage of maturation based on CD44/FSC, using Mitosox Red superoxide indicator. Right –100x micrographs of May-Grunwald/Giemsa-stained orthochromatic erythroblasts isolated using the gating employed for the Mitosox assays. (E) Top – ethidium bromide staining of the indicated mitochondrial DNA (mtDNA) amplification products, resolved on agarose gels, is shown. The mtDNA templates were obtained from Ter119+ bone marrow cells isolated from two mice of each of the indicated genotypes. Bottom – quantification of Ter119+ cell mtDNA amplification for three pairs of mice, performed using PicoGreen reagent, expressed relative to wild-type mtDNA amplification. Derivation of the calculation of for the increase in mtDNA lesions per 10kb is previously described, 26 using normalization to 117 base-pair (bp) mtDNA amplification product for mitochondrial copy number.

    Journal: Oncogene

    Article Title: Fbw7-dependent cyclin E regulation ensures terminal maturation of bone marrow erythroid cells by restraining oxidative metabolism

    doi: 10.1038/onc.2013.289

    Figure Lengend Snippet: Oxidative metabolism and mitochondrial ROS are increased in cyclin E T74A T393A erythroid cells (A) Oxidative consumption rates coupled to mitochondrial ATP production were assayed in primary Ter119+ cells from three sets of mice of the indicated genotypes using a Seahorse Extracellular Flux Analyzer. (wt= wild-type, T74A T393A= cyclin E T74A T393A cells; ** - 4×10 5 , * - 3×10 5 cells analyzed in independent assays). Error bars indicate standard error with four replicates per sample. (B) Expression of the indicated genes in purified Ter119+ CD71-high erythroid cells was measured by RT-PCR. Each grey bar represents averaged value of triplicate assays from a single knock-in mouse, expressed relative to expression in cells from age- and sex-matched wild-type controls. Inset shows total and phospho-Rb expression from similarly isolated cells primary erythroid cells. (C) Left column - bone marrow Ter119+ cells were subdivided into distinct morphologic subpopulations (II–V) based on CD71 expression vs. forward scatter (FSC) 14 , with relative abundances indicated. Right columns - intracellular reactive oxygen species were measured in the indicated erythroid cell subsets using cell-permeable 2′,7′-dichlorodihydrofluorescein diacetate (H 2 DCFDA), enabling detection by flow cytometry. Comparisons between mean fluorescence signals for bone marrow erythroid cells of four mice from each of the indicated genotype groups were made and p-values calculated using paired t-tests. (D) Left – Representative comparison is shown of mitochondrial superoxide levels measured in bone marrow erythroid cells from three pairs of mice at the indicated stage of maturation based on CD44/FSC, using Mitosox Red superoxide indicator. Right –100x micrographs of May-Grunwald/Giemsa-stained orthochromatic erythroblasts isolated using the gating employed for the Mitosox assays. (E) Top – ethidium bromide staining of the indicated mitochondrial DNA (mtDNA) amplification products, resolved on agarose gels, is shown. The mtDNA templates were obtained from Ter119+ bone marrow cells isolated from two mice of each of the indicated genotypes. Bottom – quantification of Ter119+ cell mtDNA amplification for three pairs of mice, performed using PicoGreen reagent, expressed relative to wild-type mtDNA amplification. Derivation of the calculation of for the increase in mtDNA lesions per 10kb is previously described, 26 using normalization to 117 base-pair (bp) mtDNA amplification product for mitochondrial copy number.

    Article Snippet: For separation of Ter119+ cells from whole bone marrow, cells were incubated with PE-Ter119 antibody then immunomagnetic beads (PE Selection Kit, Stem Cell Technology).

    Techniques: Mouse Assay, Expressing, Purification, Reverse Transcription Polymerase Chain Reaction, Knock-In, Isolation, Flow Cytometry, Cytometry, Fluorescence, Staining, Amplification

    Cyclin E protein regulation during terminal erythroid maturation is phosphorylation dependent (A) Left - bone marrow cells were sorted based upon expression of CD71 and Ter119 and subpopulations collected for immunoblot and RNA analyses as shown in subsequent panels. Right - lysates were prepared from the indicated bone marrow erythroid cells pooled from two wild-type mice and immunoblotted as shown. HDAC2 expression is used as a nuclear protein control for comparing the relative abundance of the cyclin E nucleoprotein in the different erythroid cell subpopulations, given the presence of enucleated cells within the R4 gate. (B) Bone marrow erythroid cells were isolated based on CD44 vs. Ter119 expression or CD44 expression vs. forward scatter (FSC) within the Ter119-positive subset, 14 with relative abundances indicated for a representative, age- and sex-matched pair. (C) Cyclin E protein levels were determined in sorted CD44/Ter119 or CD44/FSC erythroid cell subsets shown in (B) by immunoblot (top) and quantified relative to HDAC2 (bottom).

    Journal: Oncogene

    Article Title: Fbw7-dependent cyclin E regulation ensures terminal maturation of bone marrow erythroid cells by restraining oxidative metabolism

    doi: 10.1038/onc.2013.289

    Figure Lengend Snippet: Cyclin E protein regulation during terminal erythroid maturation is phosphorylation dependent (A) Left - bone marrow cells were sorted based upon expression of CD71 and Ter119 and subpopulations collected for immunoblot and RNA analyses as shown in subsequent panels. Right - lysates were prepared from the indicated bone marrow erythroid cells pooled from two wild-type mice and immunoblotted as shown. HDAC2 expression is used as a nuclear protein control for comparing the relative abundance of the cyclin E nucleoprotein in the different erythroid cell subpopulations, given the presence of enucleated cells within the R4 gate. (B) Bone marrow erythroid cells were isolated based on CD44 vs. Ter119 expression or CD44 expression vs. forward scatter (FSC) within the Ter119-positive subset, 14 with relative abundances indicated for a representative, age- and sex-matched pair. (C) Cyclin E protein levels were determined in sorted CD44/Ter119 or CD44/FSC erythroid cell subsets shown in (B) by immunoblot (top) and quantified relative to HDAC2 (bottom).

    Article Snippet: For separation of Ter119+ cells from whole bone marrow, cells were incubated with PE-Ter119 antibody then immunomagnetic beads (PE Selection Kit, Stem Cell Technology).

    Techniques: Expressing, Mouse Assay, Isolation