lc3a b alexa fluor 594 conjugate  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc lc3a b alexa fluor 594 conjugate
    (A) Expression of Atg7 , Atg5 , <t>LC3a</t> , LC3b and beclin1 tested by Q-PCR. (B) Image flow cytometric analysis of the colocalization of DAPI, LC3 and LAMP1 was performed with an Amnis ImageStream Image Flow Cytometer. The Pearson correlation coefficient of LC3 and LAMP1 colocalization was calculated. ( C ) In vivo mouse treatment with rapamycin promoted erythropoiesis. Left, peripheral blood count from different treatments. Middle, Bone marrow erythrocyte count by flow cytometry. Right, MEP cells in bone marrow were measured on a polychromatic flow cell analyzer. ( D ) In vivo mouse treatment with 3-MA prevented fasting-promoted erythropoiesis. Left, peripheral blood count from different treatments. Middle, Bone marrow erythrocyte count by flow cytometry. Right, Flow cytometric determination of developmental stages in a bone marrow erythrocytic series using fluorescent antibodies against Ter119 and CD71. I: proerythroblast (Ter119 med CD71 hi ); II: basophilic erythroblast (Ter119 hi CD71 hi ); III: polychromatophilic erythroblast (Ter119 hi CD71 med ); IV: orthochromatic erythroblast (Ter119 hi CD71 − ). (E) Confirmation of autophagy defects in two autophagy-defective mouse models with Atg7 or Atg5 deleted in hematopoietic cells. Left, Western blotting of autophagy marker proteins. Right, Image flow cytometric analysis of the colocalization of autophagy marker proteins. (F) Hematopoietic cell counts in the different treatment autophagy-defective mouse groups. Left, peripheral blood cells by routine counting. C57 + Feeding (n=11); C57 + Fasting (n=10); Atg7 −/− + Feeding (n=18); Atg7 −/− + Fasting (n=8); Atg5 −/− + Feeding (n=9); Atg5 −/− + Fasting (n=3). Middle, bone marrow erythrocytes measured on polychromatic flow cell analyzer. C57 + Feeding (n=16); C57 + Fasting (n=11); Atg7 −/− + Feeding (n=16); Atg7 −/− + Fasting (n=5); Atg5 −/− + Feeding (n=5); Atg5 −/− + Fasting (n=3). Right, Bone marrow MEP cells measured on polychromatic flow cell analyzer. C57 + Feeding (n=14); C57 + Fasting (n=11); Atg7 −/− + Feeding (n=9); Atg7 −/− + Fasting (n=4); Atg5 −/− + Feeding (n=5); Atg5 −/− + Fasting (n=3). (G) The expression of Ms4a3 and Cdk2 in MEP cells measured by quantitative PCR. (H) MEP cell cycle measured with Ki67-AF700 and Hoechst-33342 double fluorescent staining on a polychromatic flow cell analyzer.
    Lc3a B Alexa Fluor 594 Conjugate, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lc3a b alexa fluor 594 conjugate/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    lc3a b alexa fluor 594 conjugate - by Bioz Stars, 2023-02
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    Images

    1) Product Images from "Short-term intensive fasting improves red blood cell function and rejuvenates erythropoiesis via regulating MS4A3-CDK2 module"

    Article Title: Short-term intensive fasting improves red blood cell function and rejuvenates erythropoiesis via regulating MS4A3-CDK2 module

    Journal: medRxiv

    doi: 10.1101/2022.08.18.22278875

    (A) Expression of Atg7 , Atg5 , LC3a , LC3b and beclin1 tested by Q-PCR. (B) Image flow cytometric analysis of the colocalization of DAPI, LC3 and LAMP1 was performed with an Amnis ImageStream Image Flow Cytometer. The Pearson correlation coefficient of LC3 and LAMP1 colocalization was calculated. ( C ) In vivo mouse treatment with rapamycin promoted erythropoiesis. Left, peripheral blood count from different treatments. Middle, Bone marrow erythrocyte count by flow cytometry. Right, MEP cells in bone marrow were measured on a polychromatic flow cell analyzer. ( D ) In vivo mouse treatment with 3-MA prevented fasting-promoted erythropoiesis. Left, peripheral blood count from different treatments. Middle, Bone marrow erythrocyte count by flow cytometry. Right, Flow cytometric determination of developmental stages in a bone marrow erythrocytic series using fluorescent antibodies against Ter119 and CD71. I: proerythroblast (Ter119 med CD71 hi ); II: basophilic erythroblast (Ter119 hi CD71 hi ); III: polychromatophilic erythroblast (Ter119 hi CD71 med ); IV: orthochromatic erythroblast (Ter119 hi CD71 − ). (E) Confirmation of autophagy defects in two autophagy-defective mouse models with Atg7 or Atg5 deleted in hematopoietic cells. Left, Western blotting of autophagy marker proteins. Right, Image flow cytometric analysis of the colocalization of autophagy marker proteins. (F) Hematopoietic cell counts in the different treatment autophagy-defective mouse groups. Left, peripheral blood cells by routine counting. C57 + Feeding (n=11); C57 + Fasting (n=10); Atg7 −/− + Feeding (n=18); Atg7 −/− + Fasting (n=8); Atg5 −/− + Feeding (n=9); Atg5 −/− + Fasting (n=3). Middle, bone marrow erythrocytes measured on polychromatic flow cell analyzer. C57 + Feeding (n=16); C57 + Fasting (n=11); Atg7 −/− + Feeding (n=16); Atg7 −/− + Fasting (n=5); Atg5 −/− + Feeding (n=5); Atg5 −/− + Fasting (n=3). Right, Bone marrow MEP cells measured on polychromatic flow cell analyzer. C57 + Feeding (n=14); C57 + Fasting (n=11); Atg7 −/− + Feeding (n=9); Atg7 −/− + Fasting (n=4); Atg5 −/− + Feeding (n=5); Atg5 −/− + Fasting (n=3). (G) The expression of Ms4a3 and Cdk2 in MEP cells measured by quantitative PCR. (H) MEP cell cycle measured with Ki67-AF700 and Hoechst-33342 double fluorescent staining on a polychromatic flow cell analyzer.
    Figure Legend Snippet: (A) Expression of Atg7 , Atg5 , LC3a , LC3b and beclin1 tested by Q-PCR. (B) Image flow cytometric analysis of the colocalization of DAPI, LC3 and LAMP1 was performed with an Amnis ImageStream Image Flow Cytometer. The Pearson correlation coefficient of LC3 and LAMP1 colocalization was calculated. ( C ) In vivo mouse treatment with rapamycin promoted erythropoiesis. Left, peripheral blood count from different treatments. Middle, Bone marrow erythrocyte count by flow cytometry. Right, MEP cells in bone marrow were measured on a polychromatic flow cell analyzer. ( D ) In vivo mouse treatment with 3-MA prevented fasting-promoted erythropoiesis. Left, peripheral blood count from different treatments. Middle, Bone marrow erythrocyte count by flow cytometry. Right, Flow cytometric determination of developmental stages in a bone marrow erythrocytic series using fluorescent antibodies against Ter119 and CD71. I: proerythroblast (Ter119 med CD71 hi ); II: basophilic erythroblast (Ter119 hi CD71 hi ); III: polychromatophilic erythroblast (Ter119 hi CD71 med ); IV: orthochromatic erythroblast (Ter119 hi CD71 − ). (E) Confirmation of autophagy defects in two autophagy-defective mouse models with Atg7 or Atg5 deleted in hematopoietic cells. Left, Western blotting of autophagy marker proteins. Right, Image flow cytometric analysis of the colocalization of autophagy marker proteins. (F) Hematopoietic cell counts in the different treatment autophagy-defective mouse groups. Left, peripheral blood cells by routine counting. C57 + Feeding (n=11); C57 + Fasting (n=10); Atg7 −/− + Feeding (n=18); Atg7 −/− + Fasting (n=8); Atg5 −/− + Feeding (n=9); Atg5 −/− + Fasting (n=3). Middle, bone marrow erythrocytes measured on polychromatic flow cell analyzer. C57 + Feeding (n=16); C57 + Fasting (n=11); Atg7 −/− + Feeding (n=16); Atg7 −/− + Fasting (n=5); Atg5 −/− + Feeding (n=5); Atg5 −/− + Fasting (n=3). Right, Bone marrow MEP cells measured on polychromatic flow cell analyzer. C57 + Feeding (n=14); C57 + Fasting (n=11); Atg7 −/− + Feeding (n=9); Atg7 −/− + Fasting (n=4); Atg5 −/− + Feeding (n=5); Atg5 −/− + Fasting (n=3). (G) The expression of Ms4a3 and Cdk2 in MEP cells measured by quantitative PCR. (H) MEP cell cycle measured with Ki67-AF700 and Hoechst-33342 double fluorescent staining on a polychromatic flow cell analyzer.

    Techniques Used: Expressing, Flow Cytometry, In Vivo, Western Blot, Marker, Real-time Polymerase Chain Reaction, Staining

    (A) Fasting activates in vivo autophagy activity in mice. Confocal observation of autophagy markers in MEPs from Ad libitum and 6-hour-fasting mice by confocal microscopy. (B-C) Rapamycin activates in vivo basal autophagic activity in mice (n=4/group). Expression of Atg 7 , LC3a , LC3b and Beclin1 of MEP cells in mice with or without Rapamycin injection tested by Q-PCR (B). Autophagic markers of BM-MNCs from wild-type mice that received intraperitoneal injection of rapamycin were measured by Western blotting (C). (D-E) 3-MA inhibits in vivo basal autophagic activity in mice (n=4/group). Autophagic markers of BM-MNCs from wild-type mice that received intraperitoneal injection of 3-MA were measured by Q-PCR (D) and Western blotting (E).
    Figure Legend Snippet: (A) Fasting activates in vivo autophagy activity in mice. Confocal observation of autophagy markers in MEPs from Ad libitum and 6-hour-fasting mice by confocal microscopy. (B-C) Rapamycin activates in vivo basal autophagic activity in mice (n=4/group). Expression of Atg 7 , LC3a , LC3b and Beclin1 of MEP cells in mice with or without Rapamycin injection tested by Q-PCR (B). Autophagic markers of BM-MNCs from wild-type mice that received intraperitoneal injection of rapamycin were measured by Western blotting (C). (D-E) 3-MA inhibits in vivo basal autophagic activity in mice (n=4/group). Autophagic markers of BM-MNCs from wild-type mice that received intraperitoneal injection of 3-MA were measured by Q-PCR (D) and Western blotting (E).

    Techniques Used: In Vivo, Activity Assay, Confocal Microscopy, Expressing, Injection, Western Blot

    lc3a b alexa fluor 594 conjugate  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc lc3a b alexa fluor 594 conjugate
    (A) Expression of Atg7 , Atg5 , <t>LC3a</t> , LC3b and beclin1 tested by Q-PCR. (B) Image flow cytometric analysis of the colocalization of DAPI, LC3 and LAMP1 was performed with an Amnis ImageStream Image Flow Cytometer. The Pearson correlation coefficient of LC3 and LAMP1 colocalization was calculated. ( C ) In vivo mouse treatment with rapamycin promoted erythropoiesis. Left, peripheral blood count from different treatments. Middle, Bone marrow erythrocyte count by flow cytometry. Right, MEP cells in bone marrow were measured on a polychromatic flow cell analyzer. ( D ) In vivo mouse treatment with 3-MA prevented fasting-promoted erythropoiesis. Left, peripheral blood count from different treatments. Middle, Bone marrow erythrocyte count by flow cytometry. Right, Flow cytometric determination of developmental stages in a bone marrow erythrocytic series using fluorescent antibodies against Ter119 and CD71. I: proerythroblast (Ter119 med CD71 hi ); II: basophilic erythroblast (Ter119 hi CD71 hi ); III: polychromatophilic erythroblast (Ter119 hi CD71 med ); IV: orthochromatic erythroblast (Ter119 hi CD71 − ). (E) Confirmation of autophagy defects in two autophagy-defective mouse models with Atg7 or Atg5 deleted in hematopoietic cells. Left, Western blotting of autophagy marker proteins. Right, Image flow cytometric analysis of the colocalization of autophagy marker proteins. (F) Hematopoietic cell counts in the different treatment autophagy-defective mouse groups. Left, peripheral blood cells by routine counting. C57 + Feeding (n=11); C57 + Fasting (n=10); Atg7 −/− + Feeding (n=18); Atg7 −/− + Fasting (n=8); Atg5 −/− + Feeding (n=9); Atg5 −/− + Fasting (n=3). Middle, bone marrow erythrocytes measured on polychromatic flow cell analyzer. C57 + Feeding (n=16); C57 + Fasting (n=11); Atg7 −/− + Feeding (n=16); Atg7 −/− + Fasting (n=5); Atg5 −/− + Feeding (n=5); Atg5 −/− + Fasting (n=3). Right, Bone marrow MEP cells measured on polychromatic flow cell analyzer. C57 + Feeding (n=14); C57 + Fasting (n=11); Atg7 −/− + Feeding (n=9); Atg7 −/− + Fasting (n=4); Atg5 −/− + Feeding (n=5); Atg5 −/− + Fasting (n=3). (G) The expression of Ms4a3 and Cdk2 in MEP cells measured by quantitative PCR. (H) MEP cell cycle measured with Ki67-AF700 and Hoechst-33342 double fluorescent staining on a polychromatic flow cell analyzer.
    Lc3a B Alexa Fluor 594 Conjugate, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lc3a b alexa fluor 594 conjugate/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    lc3a b alexa fluor 594 conjugate - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "Short-term intensive fasting improves red blood cell function and rejuvenates erythropoiesis via regulating MS4A3-CDK2 module"

    Article Title: Short-term intensive fasting improves red blood cell function and rejuvenates erythropoiesis via regulating MS4A3-CDK2 module

    Journal: medRxiv

    doi: 10.1101/2022.08.18.22278875

    (A) Expression of Atg7 , Atg5 , LC3a , LC3b and beclin1 tested by Q-PCR. (B) Image flow cytometric analysis of the colocalization of DAPI, LC3 and LAMP1 was performed with an Amnis ImageStream Image Flow Cytometer. The Pearson correlation coefficient of LC3 and LAMP1 colocalization was calculated. ( C ) In vivo mouse treatment with rapamycin promoted erythropoiesis. Left, peripheral blood count from different treatments. Middle, Bone marrow erythrocyte count by flow cytometry. Right, MEP cells in bone marrow were measured on a polychromatic flow cell analyzer. ( D ) In vivo mouse treatment with 3-MA prevented fasting-promoted erythropoiesis. Left, peripheral blood count from different treatments. Middle, Bone marrow erythrocyte count by flow cytometry. Right, Flow cytometric determination of developmental stages in a bone marrow erythrocytic series using fluorescent antibodies against Ter119 and CD71. I: proerythroblast (Ter119 med CD71 hi ); II: basophilic erythroblast (Ter119 hi CD71 hi ); III: polychromatophilic erythroblast (Ter119 hi CD71 med ); IV: orthochromatic erythroblast (Ter119 hi CD71 − ). (E) Confirmation of autophagy defects in two autophagy-defective mouse models with Atg7 or Atg5 deleted in hematopoietic cells. Left, Western blotting of autophagy marker proteins. Right, Image flow cytometric analysis of the colocalization of autophagy marker proteins. (F) Hematopoietic cell counts in the different treatment autophagy-defective mouse groups. Left, peripheral blood cells by routine counting. C57 + Feeding (n=11); C57 + Fasting (n=10); Atg7 −/− + Feeding (n=18); Atg7 −/− + Fasting (n=8); Atg5 −/− + Feeding (n=9); Atg5 −/− + Fasting (n=3). Middle, bone marrow erythrocytes measured on polychromatic flow cell analyzer. C57 + Feeding (n=16); C57 + Fasting (n=11); Atg7 −/− + Feeding (n=16); Atg7 −/− + Fasting (n=5); Atg5 −/− + Feeding (n=5); Atg5 −/− + Fasting (n=3). Right, Bone marrow MEP cells measured on polychromatic flow cell analyzer. C57 + Feeding (n=14); C57 + Fasting (n=11); Atg7 −/− + Feeding (n=9); Atg7 −/− + Fasting (n=4); Atg5 −/− + Feeding (n=5); Atg5 −/− + Fasting (n=3). (G) The expression of Ms4a3 and Cdk2 in MEP cells measured by quantitative PCR. (H) MEP cell cycle measured with Ki67-AF700 and Hoechst-33342 double fluorescent staining on a polychromatic flow cell analyzer.
    Figure Legend Snippet: (A) Expression of Atg7 , Atg5 , LC3a , LC3b and beclin1 tested by Q-PCR. (B) Image flow cytometric analysis of the colocalization of DAPI, LC3 and LAMP1 was performed with an Amnis ImageStream Image Flow Cytometer. The Pearson correlation coefficient of LC3 and LAMP1 colocalization was calculated. ( C ) In vivo mouse treatment with rapamycin promoted erythropoiesis. Left, peripheral blood count from different treatments. Middle, Bone marrow erythrocyte count by flow cytometry. Right, MEP cells in bone marrow were measured on a polychromatic flow cell analyzer. ( D ) In vivo mouse treatment with 3-MA prevented fasting-promoted erythropoiesis. Left, peripheral blood count from different treatments. Middle, Bone marrow erythrocyte count by flow cytometry. Right, Flow cytometric determination of developmental stages in a bone marrow erythrocytic series using fluorescent antibodies against Ter119 and CD71. I: proerythroblast (Ter119 med CD71 hi ); II: basophilic erythroblast (Ter119 hi CD71 hi ); III: polychromatophilic erythroblast (Ter119 hi CD71 med ); IV: orthochromatic erythroblast (Ter119 hi CD71 − ). (E) Confirmation of autophagy defects in two autophagy-defective mouse models with Atg7 or Atg5 deleted in hematopoietic cells. Left, Western blotting of autophagy marker proteins. Right, Image flow cytometric analysis of the colocalization of autophagy marker proteins. (F) Hematopoietic cell counts in the different treatment autophagy-defective mouse groups. Left, peripheral blood cells by routine counting. C57 + Feeding (n=11); C57 + Fasting (n=10); Atg7 −/− + Feeding (n=18); Atg7 −/− + Fasting (n=8); Atg5 −/− + Feeding (n=9); Atg5 −/− + Fasting (n=3). Middle, bone marrow erythrocytes measured on polychromatic flow cell analyzer. C57 + Feeding (n=16); C57 + Fasting (n=11); Atg7 −/− + Feeding (n=16); Atg7 −/− + Fasting (n=5); Atg5 −/− + Feeding (n=5); Atg5 −/− + Fasting (n=3). Right, Bone marrow MEP cells measured on polychromatic flow cell analyzer. C57 + Feeding (n=14); C57 + Fasting (n=11); Atg7 −/− + Feeding (n=9); Atg7 −/− + Fasting (n=4); Atg5 −/− + Feeding (n=5); Atg5 −/− + Fasting (n=3). (G) The expression of Ms4a3 and Cdk2 in MEP cells measured by quantitative PCR. (H) MEP cell cycle measured with Ki67-AF700 and Hoechst-33342 double fluorescent staining on a polychromatic flow cell analyzer.

    Techniques Used: Expressing, Flow Cytometry, In Vivo, Western Blot, Marker, Real-time Polymerase Chain Reaction, Staining

    (A) Fasting activates in vivo autophagy activity in mice. Confocal observation of autophagy markers in MEPs from Ad libitum and 6-hour-fasting mice by confocal microscopy. (B-C) Rapamycin activates in vivo basal autophagic activity in mice (n=4/group). Expression of Atg 7 , LC3a , LC3b and Beclin1 of MEP cells in mice with or without Rapamycin injection tested by Q-PCR (B). Autophagic markers of BM-MNCs from wild-type mice that received intraperitoneal injection of rapamycin were measured by Western blotting (C). (D-E) 3-MA inhibits in vivo basal autophagic activity in mice (n=4/group). Autophagic markers of BM-MNCs from wild-type mice that received intraperitoneal injection of 3-MA were measured by Q-PCR (D) and Western blotting (E).
    Figure Legend Snippet: (A) Fasting activates in vivo autophagy activity in mice. Confocal observation of autophagy markers in MEPs from Ad libitum and 6-hour-fasting mice by confocal microscopy. (B-C) Rapamycin activates in vivo basal autophagic activity in mice (n=4/group). Expression of Atg 7 , LC3a , LC3b and Beclin1 of MEP cells in mice with or without Rapamycin injection tested by Q-PCR (B). Autophagic markers of BM-MNCs from wild-type mice that received intraperitoneal injection of rapamycin were measured by Western blotting (C). (D-E) 3-MA inhibits in vivo basal autophagic activity in mice (n=4/group). Autophagic markers of BM-MNCs from wild-type mice that received intraperitoneal injection of 3-MA were measured by Q-PCR (D) and Western blotting (E).

    Techniques Used: In Vivo, Activity Assay, Confocal Microscopy, Expressing, Injection, Western Blot

    rabbit catalase antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit catalase antibody
    Rabbit Catalase Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit catalase antibody/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
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    lc3  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc lc3
    Reduction of autophagy is associated with aged hematopoiesis in human population. (a) Linear regression and Pearson correlation coefficients of peripheral blood counts in aging human population. A pool of peripheral blood count information from physical examination of 4250 people aged from 20 to 90 years was analyzed using SPSS statistic software. (b) Quantitative PCR measuring the expression of autophagy‐essential genes in human bone marrow primary HSC‐enriched hematopoietic cells (CD45CD34) from the indicated two age groups. (c, d) Quantitative ImageStream detection of basal autophagy levels in human bone marrow primary hematopoietic cells (CD45), HSC‐enriched hematopoietic cells (CD45CD34) from the two age groups. Left, statistical data from individual human samples. Right, representative images of the cells, either single‐stained (CD45—blue; CD34—purple; <t>LC3—green;</t> Lamp1—red) or stained for both markers (merge of LC3 and Lamp1). Bar, 10 μm
    Lc3, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lc3/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    lc3 - by Bioz Stars, 2023-02
    94/100 stars

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    1) Product Images from "Autophagy‐Sirt3 axis decelerates hematopoietic aging"

    Article Title: Autophagy‐Sirt3 axis decelerates hematopoietic aging

    Journal: Aging Cell

    doi: 10.1111/acel.13232

    Reduction of autophagy is associated with aged hematopoiesis in human population. (a) Linear regression and Pearson correlation coefficients of peripheral blood counts in aging human population. A pool of peripheral blood count information from physical examination of 4250 people aged from 20 to 90 years was analyzed using SPSS statistic software. (b) Quantitative PCR measuring the expression of autophagy‐essential genes in human bone marrow primary HSC‐enriched hematopoietic cells (CD45CD34) from the indicated two age groups. (c, d) Quantitative ImageStream detection of basal autophagy levels in human bone marrow primary hematopoietic cells (CD45), HSC‐enriched hematopoietic cells (CD45CD34) from the two age groups. Left, statistical data from individual human samples. Right, representative images of the cells, either single‐stained (CD45—blue; CD34—purple; LC3—green; Lamp1—red) or stained for both markers (merge of LC3 and Lamp1). Bar, 10 μm
    Figure Legend Snippet: Reduction of autophagy is associated with aged hematopoiesis in human population. (a) Linear regression and Pearson correlation coefficients of peripheral blood counts in aging human population. A pool of peripheral blood count information from physical examination of 4250 people aged from 20 to 90 years was analyzed using SPSS statistic software. (b) Quantitative PCR measuring the expression of autophagy‐essential genes in human bone marrow primary HSC‐enriched hematopoietic cells (CD45CD34) from the indicated two age groups. (c, d) Quantitative ImageStream detection of basal autophagy levels in human bone marrow primary hematopoietic cells (CD45), HSC‐enriched hematopoietic cells (CD45CD34) from the two age groups. Left, statistical data from individual human samples. Right, representative images of the cells, either single‐stained (CD45—blue; CD34—purple; LC3—green; Lamp1—red) or stained for both markers (merge of LC3 and Lamp1). Bar, 10 μm

    Techniques Used: Software, Real-time Polymerase Chain Reaction, Expressing, Staining

    Sirt3 expression depends on Atg7 in the mouse bone marrow HSC‐enriched hematopoietic cells. (a) The volcano map of differentially expressed genes in Atg7 −/− HSC‐enriched hematopoietic cells as compared to Atg7 +/+ HSC‐enriched cells. A total of 1062 genes were significantly upregulated, while 789 genes were downregulated in Atg7‐depleted HSC‐enriched cells. (b) RNA sequencing reveals Sirt3 dependency on Atg7. Left, the heatmap of Sirtuin family between Atg7 +/+ and Atg7 −/− HSC‐enriched cells. The result shows that only Sirt3 expression is reduced in the HSPCs of all of the three Atg7 −/− mice. Middle, the reduction levels of the expression of seven Sirtuin family members in the HSC‐enriched cells due to Atg7 deletion. The result shows that only Sirt3 expression is significantly reduced in the Atg7 −/− HSC‐enriched cells. Right, the relative expression levels of Sirtuin family members in wild‐type HSC‐enriched cells. The result shows that Sirt3 is the highest expressed member in Sirtuin family in the HSC‐enriched cells of the wild‐type mice. (c) Measurement of expression levels of the Sirtuin family members by quantitative PCR in the Atg7 +/+ and Atg7 −/− HSC‐enriched cells. (d) Measurement of Sirt3 pre‐mRNA by quantitative PCR. pre‐mRNA expression was normalized to gapdh level. (e) Flow cytometric examination of Sirt3 protein expression in HSC‐enriched cells, hematopoietic progenitor cells, and terminally differentiated hematopoietic cells of wild‐type and Atg7‐deleted mice aged 10 weeks. Left, representative data of Sirt3 protein expression levels in HSC‐enriched cells (LSK), HPCs (Lin − ), and terminally differentiated hematopoietic cells (Lin + ) by flow cytometry. Right, statistic data for comparison of Sirt3 expression levels. (f) Measurement of expression of Sirt3, Atg7, and LC3 proteins by Western blotting in Atg7 +/+ and Atg7 −/− bone marrow mononuclear cells (MNCs) and hematopoietic progenitor cells (Lin − sorted) from 10‐week‐old mice. Gapdh serves as a loading control. (g) Confocal detection of Sirt3 protein in the Atg7 +/+ and Atg7 −/− HSC‐enriched cells. Representative images were taken from bone marrow HSC‐enriched cells of mice aged at 2 weeks and 10 weeks. (h) Quantitative PCR measurement of Sirt3 expression levels in the HSC‐enriched cells from aging wild‐type mice. (i) Time course comparison on Sirt3 expression by quantitative PCR in HSC‐enriched cells of wild‐type and Atg7‐deleted mice
    Figure Legend Snippet: Sirt3 expression depends on Atg7 in the mouse bone marrow HSC‐enriched hematopoietic cells. (a) The volcano map of differentially expressed genes in Atg7 −/− HSC‐enriched hematopoietic cells as compared to Atg7 +/+ HSC‐enriched cells. A total of 1062 genes were significantly upregulated, while 789 genes were downregulated in Atg7‐depleted HSC‐enriched cells. (b) RNA sequencing reveals Sirt3 dependency on Atg7. Left, the heatmap of Sirtuin family between Atg7 +/+ and Atg7 −/− HSC‐enriched cells. The result shows that only Sirt3 expression is reduced in the HSPCs of all of the three Atg7 −/− mice. Middle, the reduction levels of the expression of seven Sirtuin family members in the HSC‐enriched cells due to Atg7 deletion. The result shows that only Sirt3 expression is significantly reduced in the Atg7 −/− HSC‐enriched cells. Right, the relative expression levels of Sirtuin family members in wild‐type HSC‐enriched cells. The result shows that Sirt3 is the highest expressed member in Sirtuin family in the HSC‐enriched cells of the wild‐type mice. (c) Measurement of expression levels of the Sirtuin family members by quantitative PCR in the Atg7 +/+ and Atg7 −/− HSC‐enriched cells. (d) Measurement of Sirt3 pre‐mRNA by quantitative PCR. pre‐mRNA expression was normalized to gapdh level. (e) Flow cytometric examination of Sirt3 protein expression in HSC‐enriched cells, hematopoietic progenitor cells, and terminally differentiated hematopoietic cells of wild‐type and Atg7‐deleted mice aged 10 weeks. Left, representative data of Sirt3 protein expression levels in HSC‐enriched cells (LSK), HPCs (Lin − ), and terminally differentiated hematopoietic cells (Lin + ) by flow cytometry. Right, statistic data for comparison of Sirt3 expression levels. (f) Measurement of expression of Sirt3, Atg7, and LC3 proteins by Western blotting in Atg7 +/+ and Atg7 −/− bone marrow mononuclear cells (MNCs) and hematopoietic progenitor cells (Lin − sorted) from 10‐week‐old mice. Gapdh serves as a loading control. (g) Confocal detection of Sirt3 protein in the Atg7 +/+ and Atg7 −/− HSC‐enriched cells. Representative images were taken from bone marrow HSC‐enriched cells of mice aged at 2 weeks and 10 weeks. (h) Quantitative PCR measurement of Sirt3 expression levels in the HSC‐enriched cells from aging wild‐type mice. (i) Time course comparison on Sirt3 expression by quantitative PCR in HSC‐enriched cells of wild‐type and Atg7‐deleted mice

    Techniques Used: Expressing, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Flow Cytometry, Western Blot

    Enhancement of autophagy upregulates Sirt3 expression, and overexpression of Sirt3 reduces oxidative stress and rescues hematopoietic aging in autophagy‐defective mice. (a) Rapamycin upregulates the mammalian autophagy‐essential genes in wild‐type HSC‐enriched hematopoietic cells. Relative expression levels were measured by quantitative PCR. (b) Rapamycin increases the co‐localization between LC3 and Lamp1, an indicator for the formation of autolysosomes, in wild‐type HSC‐enriched hematopoietic cells. The representative images and statistical results were taken and analyzed by Amnis ImageStream image flow cytometer. Rapamycin 200 ng/ml, bafilomycin 10 nM. (c) Quantitative PCR detection of ex vivo autophagy‐induced transcription levels by rapamycin in primary HSC‐enriched hematopoietic cells of wild‐type and Atg7‐deleted mice for Sirt3 (left) and the rest of Sirtuin family members (right). (d) Ex vivo activation of autophagy by starvation selectively upregulates Sirt3 transcription in primary HSC‐enriched hematopoietic cells of wild‐type mice. Transcription levels for Sirtuin family members were detected by quantitative PCR. (e) In vivo activation of autophagy by progressive starvation upregulates Sirt3 transcription. Transcription levels for Sirt3 (left) and autophagy‐essential genes (right) in the HSC‐enriched hematopoietic cells from long‐term progressively starved mice were detected by quantitative PCR. Starvation was achieved by calorie restriction where the amount of feed for the mice was increasingly reduced by 10% of that fed last week, for a total of 4 weeks. (f‐j) Overexpression of Sirt3 reduces oxidative stress and rescues hematopoietic aging in autophagy‐defective mice. Diagrammatic sketch showing the generation of the mouse model with in vivo ectopic overexpression of Sirt3 by lentivirus infection in mouse HSC‐enriched hematopoietic cells, followed by the infected cell transplantation (f); Western blotting analysis of Sirt3 overexpression vector in mammalian cells infected by lentivirus and quantitative result for Sirt3 overexpression in NIH3T3 cells (g); flow cytometric examination of ex vivo ROS levels in the HSC‐enriched cells (h); donor hematopoietic engraft percentage measured 8 weeks after transplantation (i) and hematopoietic reconstitution for myeloid and lymphoid lineages (j) in the Atg7 +/+ and Atg7 −/− host mice infected by control or Sirt3 overexpression lentivirus
    Figure Legend Snippet: Enhancement of autophagy upregulates Sirt3 expression, and overexpression of Sirt3 reduces oxidative stress and rescues hematopoietic aging in autophagy‐defective mice. (a) Rapamycin upregulates the mammalian autophagy‐essential genes in wild‐type HSC‐enriched hematopoietic cells. Relative expression levels were measured by quantitative PCR. (b) Rapamycin increases the co‐localization between LC3 and Lamp1, an indicator for the formation of autolysosomes, in wild‐type HSC‐enriched hematopoietic cells. The representative images and statistical results were taken and analyzed by Amnis ImageStream image flow cytometer. Rapamycin 200 ng/ml, bafilomycin 10 nM. (c) Quantitative PCR detection of ex vivo autophagy‐induced transcription levels by rapamycin in primary HSC‐enriched hematopoietic cells of wild‐type and Atg7‐deleted mice for Sirt3 (left) and the rest of Sirtuin family members (right). (d) Ex vivo activation of autophagy by starvation selectively upregulates Sirt3 transcription in primary HSC‐enriched hematopoietic cells of wild‐type mice. Transcription levels for Sirtuin family members were detected by quantitative PCR. (e) In vivo activation of autophagy by progressive starvation upregulates Sirt3 transcription. Transcription levels for Sirt3 (left) and autophagy‐essential genes (right) in the HSC‐enriched hematopoietic cells from long‐term progressively starved mice were detected by quantitative PCR. Starvation was achieved by calorie restriction where the amount of feed for the mice was increasingly reduced by 10% of that fed last week, for a total of 4 weeks. (f‐j) Overexpression of Sirt3 reduces oxidative stress and rescues hematopoietic aging in autophagy‐defective mice. Diagrammatic sketch showing the generation of the mouse model with in vivo ectopic overexpression of Sirt3 by lentivirus infection in mouse HSC‐enriched hematopoietic cells, followed by the infected cell transplantation (f); Western blotting analysis of Sirt3 overexpression vector in mammalian cells infected by lentivirus and quantitative result for Sirt3 overexpression in NIH3T3 cells (g); flow cytometric examination of ex vivo ROS levels in the HSC‐enriched cells (h); donor hematopoietic engraft percentage measured 8 weeks after transplantation (i) and hematopoietic reconstitution for myeloid and lymphoid lineages (j) in the Atg7 +/+ and Atg7 −/− host mice infected by control or Sirt3 overexpression lentivirus

    Techniques Used: Expressing, Over Expression, Real-time Polymerase Chain Reaction, Flow Cytometry, Ex Vivo, Activation Assay, In Vivo, Infection, Transplantation Assay, Western Blot, Plasmid Preparation

    protein yap  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc lc3a b alexa fluor 594 conjugate
    (A) Expression of Atg7 , Atg5 , <t>LC3a</t> , LC3b and beclin1 tested by Q-PCR. (B) Image flow cytometric analysis of the colocalization of DAPI, LC3 and LAMP1 was performed with an Amnis ImageStream Image Flow Cytometer. The Pearson correlation coefficient of LC3 and LAMP1 colocalization was calculated. ( C ) In vivo mouse treatment with rapamycin promoted erythropoiesis. Left, peripheral blood count from different treatments. Middle, Bone marrow erythrocyte count by flow cytometry. Right, MEP cells in bone marrow were measured on a polychromatic flow cell analyzer. ( D ) In vivo mouse treatment with 3-MA prevented fasting-promoted erythropoiesis. Left, peripheral blood count from different treatments. Middle, Bone marrow erythrocyte count by flow cytometry. Right, Flow cytometric determination of developmental stages in a bone marrow erythrocytic series using fluorescent antibodies against Ter119 and CD71. I: proerythroblast (Ter119 med CD71 hi ); II: basophilic erythroblast (Ter119 hi CD71 hi ); III: polychromatophilic erythroblast (Ter119 hi CD71 med ); IV: orthochromatic erythroblast (Ter119 hi CD71 − ). (E) Confirmation of autophagy defects in two autophagy-defective mouse models with Atg7 or Atg5 deleted in hematopoietic cells. Left, Western blotting of autophagy marker proteins. Right, Image flow cytometric analysis of the colocalization of autophagy marker proteins. (F) Hematopoietic cell counts in the different treatment autophagy-defective mouse groups. Left, peripheral blood cells by routine counting. C57 + Feeding (n=11); C57 + Fasting (n=10); Atg7 −/− + Feeding (n=18); Atg7 −/− + Fasting (n=8); Atg5 −/− + Feeding (n=9); Atg5 −/− + Fasting (n=3). Middle, bone marrow erythrocytes measured on polychromatic flow cell analyzer. C57 + Feeding (n=16); C57 + Fasting (n=11); Atg7 −/− + Feeding (n=16); Atg7 −/− + Fasting (n=5); Atg5 −/− + Feeding (n=5); Atg5 −/− + Fasting (n=3). Right, Bone marrow MEP cells measured on polychromatic flow cell analyzer. C57 + Feeding (n=14); C57 + Fasting (n=11); Atg7 −/− + Feeding (n=9); Atg7 −/− + Fasting (n=4); Atg5 −/− + Feeding (n=5); Atg5 −/− + Fasting (n=3). (G) The expression of Ms4a3 and Cdk2 in MEP cells measured by quantitative PCR. (H) MEP cell cycle measured with Ki67-AF700 and Hoechst-33342 double fluorescent staining on a polychromatic flow cell analyzer.
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    (A) Expression of Atg7 , Atg5 , <t>LC3a</t> , LC3b and beclin1 tested by Q-PCR. (B) Image flow cytometric analysis of the colocalization of DAPI, LC3 and LAMP1 was performed with an Amnis ImageStream Image Flow Cytometer. The Pearson correlation coefficient of LC3 and LAMP1 colocalization was calculated. ( C ) In vivo mouse treatment with rapamycin promoted erythropoiesis. Left, peripheral blood count from different treatments. Middle, Bone marrow erythrocyte count by flow cytometry. Right, MEP cells in bone marrow were measured on a polychromatic flow cell analyzer. ( D ) In vivo mouse treatment with 3-MA prevented fasting-promoted erythropoiesis. Left, peripheral blood count from different treatments. Middle, Bone marrow erythrocyte count by flow cytometry. Right, Flow cytometric determination of developmental stages in a bone marrow erythrocytic series using fluorescent antibodies against Ter119 and CD71. I: proerythroblast (Ter119 med CD71 hi ); II: basophilic erythroblast (Ter119 hi CD71 hi ); III: polychromatophilic erythroblast (Ter119 hi CD71 med ); IV: orthochromatic erythroblast (Ter119 hi CD71 − ). (E) Confirmation of autophagy defects in two autophagy-defective mouse models with Atg7 or Atg5 deleted in hematopoietic cells. Left, Western blotting of autophagy marker proteins. Right, Image flow cytometric analysis of the colocalization of autophagy marker proteins. (F) Hematopoietic cell counts in the different treatment autophagy-defective mouse groups. Left, peripheral blood cells by routine counting. C57 + Feeding (n=11); C57 + Fasting (n=10); Atg7 −/− + Feeding (n=18); Atg7 −/− + Fasting (n=8); Atg5 −/− + Feeding (n=9); Atg5 −/− + Fasting (n=3). Middle, bone marrow erythrocytes measured on polychromatic flow cell analyzer. C57 + Feeding (n=16); C57 + Fasting (n=11); Atg7 −/− + Feeding (n=16); Atg7 −/− + Fasting (n=5); Atg5 −/− + Feeding (n=5); Atg5 −/− + Fasting (n=3). Right, Bone marrow MEP cells measured on polychromatic flow cell analyzer. C57 + Feeding (n=14); C57 + Fasting (n=11); Atg7 −/− + Feeding (n=9); Atg7 −/− + Fasting (n=4); Atg5 −/− + Feeding (n=5); Atg5 −/− + Fasting (n=3). (G) The expression of Ms4a3 and Cdk2 in MEP cells measured by quantitative PCR. (H) MEP cell cycle measured with Ki67-AF700 and Hoechst-33342 double fluorescent staining on a polychromatic flow cell analyzer.
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    Reduction of autophagy is associated with aged hematopoiesis in human population. (a) Linear regression and Pearson correlation coefficients of peripheral blood counts in aging human population. A pool of peripheral blood count information from physical examination of 4250 people aged from 20 to 90 years was analyzed using SPSS statistic software. (b) Quantitative PCR measuring the expression of autophagy‐essential genes in human bone marrow primary HSC‐enriched hematopoietic cells (CD45CD34) from the indicated two age groups. (c, d) Quantitative ImageStream detection of basal autophagy levels in human bone marrow primary hematopoietic cells (CD45), HSC‐enriched hematopoietic cells (CD45CD34) from the two age groups. Left, statistical data from individual human samples. Right, representative images of the cells, either single‐stained (CD45—blue; CD34—purple; <t>LC3—green;</t> Lamp1—red) or stained for both markers (merge of LC3 and Lamp1). Bar, 10 μm
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    (A) Expression of Atg7 , Atg5 , LC3a , LC3b and beclin1 tested by Q-PCR. (B) Image flow cytometric analysis of the colocalization of DAPI, LC3 and LAMP1 was performed with an Amnis ImageStream Image Flow Cytometer. The Pearson correlation coefficient of LC3 and LAMP1 colocalization was calculated. ( C ) In vivo mouse treatment with rapamycin promoted erythropoiesis. Left, peripheral blood count from different treatments. Middle, Bone marrow erythrocyte count by flow cytometry. Right, MEP cells in bone marrow were measured on a polychromatic flow cell analyzer. ( D ) In vivo mouse treatment with 3-MA prevented fasting-promoted erythropoiesis. Left, peripheral blood count from different treatments. Middle, Bone marrow erythrocyte count by flow cytometry. Right, Flow cytometric determination of developmental stages in a bone marrow erythrocytic series using fluorescent antibodies against Ter119 and CD71. I: proerythroblast (Ter119 med CD71 hi ); II: basophilic erythroblast (Ter119 hi CD71 hi ); III: polychromatophilic erythroblast (Ter119 hi CD71 med ); IV: orthochromatic erythroblast (Ter119 hi CD71 − ). (E) Confirmation of autophagy defects in two autophagy-defective mouse models with Atg7 or Atg5 deleted in hematopoietic cells. Left, Western blotting of autophagy marker proteins. Right, Image flow cytometric analysis of the colocalization of autophagy marker proteins. (F) Hematopoietic cell counts in the different treatment autophagy-defective mouse groups. Left, peripheral blood cells by routine counting. C57 + Feeding (n=11); C57 + Fasting (n=10); Atg7 −/− + Feeding (n=18); Atg7 −/− + Fasting (n=8); Atg5 −/− + Feeding (n=9); Atg5 −/− + Fasting (n=3). Middle, bone marrow erythrocytes measured on polychromatic flow cell analyzer. C57 + Feeding (n=16); C57 + Fasting (n=11); Atg7 −/− + Feeding (n=16); Atg7 −/− + Fasting (n=5); Atg5 −/− + Feeding (n=5); Atg5 −/− + Fasting (n=3). Right, Bone marrow MEP cells measured on polychromatic flow cell analyzer. C57 + Feeding (n=14); C57 + Fasting (n=11); Atg7 −/− + Feeding (n=9); Atg7 −/− + Fasting (n=4); Atg5 −/− + Feeding (n=5); Atg5 −/− + Fasting (n=3). (G) The expression of Ms4a3 and Cdk2 in MEP cells measured by quantitative PCR. (H) MEP cell cycle measured with Ki67-AF700 and Hoechst-33342 double fluorescent staining on a polychromatic flow cell analyzer.

    Journal: medRxiv

    Article Title: Short-term intensive fasting improves red blood cell function and rejuvenates erythropoiesis via regulating MS4A3-CDK2 module

    doi: 10.1101/2022.08.18.22278875

    Figure Lengend Snippet: (A) Expression of Atg7 , Atg5 , LC3a , LC3b and beclin1 tested by Q-PCR. (B) Image flow cytometric analysis of the colocalization of DAPI, LC3 and LAMP1 was performed with an Amnis ImageStream Image Flow Cytometer. The Pearson correlation coefficient of LC3 and LAMP1 colocalization was calculated. ( C ) In vivo mouse treatment with rapamycin promoted erythropoiesis. Left, peripheral blood count from different treatments. Middle, Bone marrow erythrocyte count by flow cytometry. Right, MEP cells in bone marrow were measured on a polychromatic flow cell analyzer. ( D ) In vivo mouse treatment with 3-MA prevented fasting-promoted erythropoiesis. Left, peripheral blood count from different treatments. Middle, Bone marrow erythrocyte count by flow cytometry. Right, Flow cytometric determination of developmental stages in a bone marrow erythrocytic series using fluorescent antibodies against Ter119 and CD71. I: proerythroblast (Ter119 med CD71 hi ); II: basophilic erythroblast (Ter119 hi CD71 hi ); III: polychromatophilic erythroblast (Ter119 hi CD71 med ); IV: orthochromatic erythroblast (Ter119 hi CD71 − ). (E) Confirmation of autophagy defects in two autophagy-defective mouse models with Atg7 or Atg5 deleted in hematopoietic cells. Left, Western blotting of autophagy marker proteins. Right, Image flow cytometric analysis of the colocalization of autophagy marker proteins. (F) Hematopoietic cell counts in the different treatment autophagy-defective mouse groups. Left, peripheral blood cells by routine counting. C57 + Feeding (n=11); C57 + Fasting (n=10); Atg7 −/− + Feeding (n=18); Atg7 −/− + Fasting (n=8); Atg5 −/− + Feeding (n=9); Atg5 −/− + Fasting (n=3). Middle, bone marrow erythrocytes measured on polychromatic flow cell analyzer. C57 + Feeding (n=16); C57 + Fasting (n=11); Atg7 −/− + Feeding (n=16); Atg7 −/− + Fasting (n=5); Atg5 −/− + Feeding (n=5); Atg5 −/− + Fasting (n=3). Right, Bone marrow MEP cells measured on polychromatic flow cell analyzer. C57 + Feeding (n=14); C57 + Fasting (n=11); Atg7 −/− + Feeding (n=9); Atg7 −/− + Fasting (n=4); Atg5 −/− + Feeding (n=5); Atg5 −/− + Fasting (n=3). (G) The expression of Ms4a3 and Cdk2 in MEP cells measured by quantitative PCR. (H) MEP cell cycle measured with Ki67-AF700 and Hoechst-33342 double fluorescent staining on a polychromatic flow cell analyzer.

    Article Snippet: Image flow cytometric analysis of colocalization of CD45-FITC (157214, Biolegend), LC3A/B Alexa Fluor® 594 Conjugate (14079s, CST) and Lamp1 (ab208943, Abcam) was performed with an Amnis ImageStream Image Flow Cytometer (Amnis, Merck Millipore).

    Techniques: Expressing, Flow Cytometry, In Vivo, Western Blot, Marker, Real-time Polymerase Chain Reaction, Staining

    (A) Fasting activates in vivo autophagy activity in mice. Confocal observation of autophagy markers in MEPs from Ad libitum and 6-hour-fasting mice by confocal microscopy. (B-C) Rapamycin activates in vivo basal autophagic activity in mice (n=4/group). Expression of Atg 7 , LC3a , LC3b and Beclin1 of MEP cells in mice with or without Rapamycin injection tested by Q-PCR (B). Autophagic markers of BM-MNCs from wild-type mice that received intraperitoneal injection of rapamycin were measured by Western blotting (C). (D-E) 3-MA inhibits in vivo basal autophagic activity in mice (n=4/group). Autophagic markers of BM-MNCs from wild-type mice that received intraperitoneal injection of 3-MA were measured by Q-PCR (D) and Western blotting (E).

    Journal: medRxiv

    Article Title: Short-term intensive fasting improves red blood cell function and rejuvenates erythropoiesis via regulating MS4A3-CDK2 module

    doi: 10.1101/2022.08.18.22278875

    Figure Lengend Snippet: (A) Fasting activates in vivo autophagy activity in mice. Confocal observation of autophagy markers in MEPs from Ad libitum and 6-hour-fasting mice by confocal microscopy. (B-C) Rapamycin activates in vivo basal autophagic activity in mice (n=4/group). Expression of Atg 7 , LC3a , LC3b and Beclin1 of MEP cells in mice with or without Rapamycin injection tested by Q-PCR (B). Autophagic markers of BM-MNCs from wild-type mice that received intraperitoneal injection of rapamycin were measured by Western blotting (C). (D-E) 3-MA inhibits in vivo basal autophagic activity in mice (n=4/group). Autophagic markers of BM-MNCs from wild-type mice that received intraperitoneal injection of 3-MA were measured by Q-PCR (D) and Western blotting (E).

    Article Snippet: Image flow cytometric analysis of colocalization of CD45-FITC (157214, Biolegend), LC3A/B Alexa Fluor® 594 Conjugate (14079s, CST) and Lamp1 (ab208943, Abcam) was performed with an Amnis ImageStream Image Flow Cytometer (Amnis, Merck Millipore).

    Techniques: In Vivo, Activity Assay, Confocal Microscopy, Expressing, Injection, Western Blot

    Reduction of autophagy is associated with aged hematopoiesis in human population. (a) Linear regression and Pearson correlation coefficients of peripheral blood counts in aging human population. A pool of peripheral blood count information from physical examination of 4250 people aged from 20 to 90 years was analyzed using SPSS statistic software. (b) Quantitative PCR measuring the expression of autophagy‐essential genes in human bone marrow primary HSC‐enriched hematopoietic cells (CD45CD34) from the indicated two age groups. (c, d) Quantitative ImageStream detection of basal autophagy levels in human bone marrow primary hematopoietic cells (CD45), HSC‐enriched hematopoietic cells (CD45CD34) from the two age groups. Left, statistical data from individual human samples. Right, representative images of the cells, either single‐stained (CD45—blue; CD34—purple; LC3—green; Lamp1—red) or stained for both markers (merge of LC3 and Lamp1). Bar, 10 μm

    Journal: Aging Cell

    Article Title: Autophagy‐Sirt3 axis decelerates hematopoietic aging

    doi: 10.1111/acel.13232

    Figure Lengend Snippet: Reduction of autophagy is associated with aged hematopoiesis in human population. (a) Linear regression and Pearson correlation coefficients of peripheral blood counts in aging human population. A pool of peripheral blood count information from physical examination of 4250 people aged from 20 to 90 years was analyzed using SPSS statistic software. (b) Quantitative PCR measuring the expression of autophagy‐essential genes in human bone marrow primary HSC‐enriched hematopoietic cells (CD45CD34) from the indicated two age groups. (c, d) Quantitative ImageStream detection of basal autophagy levels in human bone marrow primary hematopoietic cells (CD45), HSC‐enriched hematopoietic cells (CD45CD34) from the two age groups. Left, statistical data from individual human samples. Right, representative images of the cells, either single‐stained (CD45—blue; CD34—purple; LC3—green; Lamp1—red) or stained for both markers (merge of LC3 and Lamp1). Bar, 10 μm

    Article Snippet: Image flow cytometric analysis of co‐localization of CD34 (555821, BD Pharmingen), CD45 (563879, BD Pharmingen), LC3 (14079S, Cell Signaling Technology), and Lamp1 (ab24170, Abcam) was performed with Amnis ImageStream Image Flow Cytometer (Amnis, Merck Millipore).

    Techniques: Software, Real-time Polymerase Chain Reaction, Expressing, Staining

    Sirt3 expression depends on Atg7 in the mouse bone marrow HSC‐enriched hematopoietic cells. (a) The volcano map of differentially expressed genes in Atg7 −/− HSC‐enriched hematopoietic cells as compared to Atg7 +/+ HSC‐enriched cells. A total of 1062 genes were significantly upregulated, while 789 genes were downregulated in Atg7‐depleted HSC‐enriched cells. (b) RNA sequencing reveals Sirt3 dependency on Atg7. Left, the heatmap of Sirtuin family between Atg7 +/+ and Atg7 −/− HSC‐enriched cells. The result shows that only Sirt3 expression is reduced in the HSPCs of all of the three Atg7 −/− mice. Middle, the reduction levels of the expression of seven Sirtuin family members in the HSC‐enriched cells due to Atg7 deletion. The result shows that only Sirt3 expression is significantly reduced in the Atg7 −/− HSC‐enriched cells. Right, the relative expression levels of Sirtuin family members in wild‐type HSC‐enriched cells. The result shows that Sirt3 is the highest expressed member in Sirtuin family in the HSC‐enriched cells of the wild‐type mice. (c) Measurement of expression levels of the Sirtuin family members by quantitative PCR in the Atg7 +/+ and Atg7 −/− HSC‐enriched cells. (d) Measurement of Sirt3 pre‐mRNA by quantitative PCR. pre‐mRNA expression was normalized to gapdh level. (e) Flow cytometric examination of Sirt3 protein expression in HSC‐enriched cells, hematopoietic progenitor cells, and terminally differentiated hematopoietic cells of wild‐type and Atg7‐deleted mice aged 10 weeks. Left, representative data of Sirt3 protein expression levels in HSC‐enriched cells (LSK), HPCs (Lin − ), and terminally differentiated hematopoietic cells (Lin + ) by flow cytometry. Right, statistic data for comparison of Sirt3 expression levels. (f) Measurement of expression of Sirt3, Atg7, and LC3 proteins by Western blotting in Atg7 +/+ and Atg7 −/− bone marrow mononuclear cells (MNCs) and hematopoietic progenitor cells (Lin − sorted) from 10‐week‐old mice. Gapdh serves as a loading control. (g) Confocal detection of Sirt3 protein in the Atg7 +/+ and Atg7 −/− HSC‐enriched cells. Representative images were taken from bone marrow HSC‐enriched cells of mice aged at 2 weeks and 10 weeks. (h) Quantitative PCR measurement of Sirt3 expression levels in the HSC‐enriched cells from aging wild‐type mice. (i) Time course comparison on Sirt3 expression by quantitative PCR in HSC‐enriched cells of wild‐type and Atg7‐deleted mice

    Journal: Aging Cell

    Article Title: Autophagy‐Sirt3 axis decelerates hematopoietic aging

    doi: 10.1111/acel.13232

    Figure Lengend Snippet: Sirt3 expression depends on Atg7 in the mouse bone marrow HSC‐enriched hematopoietic cells. (a) The volcano map of differentially expressed genes in Atg7 −/− HSC‐enriched hematopoietic cells as compared to Atg7 +/+ HSC‐enriched cells. A total of 1062 genes were significantly upregulated, while 789 genes were downregulated in Atg7‐depleted HSC‐enriched cells. (b) RNA sequencing reveals Sirt3 dependency on Atg7. Left, the heatmap of Sirtuin family between Atg7 +/+ and Atg7 −/− HSC‐enriched cells. The result shows that only Sirt3 expression is reduced in the HSPCs of all of the three Atg7 −/− mice. Middle, the reduction levels of the expression of seven Sirtuin family members in the HSC‐enriched cells due to Atg7 deletion. The result shows that only Sirt3 expression is significantly reduced in the Atg7 −/− HSC‐enriched cells. Right, the relative expression levels of Sirtuin family members in wild‐type HSC‐enriched cells. The result shows that Sirt3 is the highest expressed member in Sirtuin family in the HSC‐enriched cells of the wild‐type mice. (c) Measurement of expression levels of the Sirtuin family members by quantitative PCR in the Atg7 +/+ and Atg7 −/− HSC‐enriched cells. (d) Measurement of Sirt3 pre‐mRNA by quantitative PCR. pre‐mRNA expression was normalized to gapdh level. (e) Flow cytometric examination of Sirt3 protein expression in HSC‐enriched cells, hematopoietic progenitor cells, and terminally differentiated hematopoietic cells of wild‐type and Atg7‐deleted mice aged 10 weeks. Left, representative data of Sirt3 protein expression levels in HSC‐enriched cells (LSK), HPCs (Lin − ), and terminally differentiated hematopoietic cells (Lin + ) by flow cytometry. Right, statistic data for comparison of Sirt3 expression levels. (f) Measurement of expression of Sirt3, Atg7, and LC3 proteins by Western blotting in Atg7 +/+ and Atg7 −/− bone marrow mononuclear cells (MNCs) and hematopoietic progenitor cells (Lin − sorted) from 10‐week‐old mice. Gapdh serves as a loading control. (g) Confocal detection of Sirt3 protein in the Atg7 +/+ and Atg7 −/− HSC‐enriched cells. Representative images were taken from bone marrow HSC‐enriched cells of mice aged at 2 weeks and 10 weeks. (h) Quantitative PCR measurement of Sirt3 expression levels in the HSC‐enriched cells from aging wild‐type mice. (i) Time course comparison on Sirt3 expression by quantitative PCR in HSC‐enriched cells of wild‐type and Atg7‐deleted mice

    Article Snippet: Image flow cytometric analysis of co‐localization of CD34 (555821, BD Pharmingen), CD45 (563879, BD Pharmingen), LC3 (14079S, Cell Signaling Technology), and Lamp1 (ab24170, Abcam) was performed with Amnis ImageStream Image Flow Cytometer (Amnis, Merck Millipore).

    Techniques: Expressing, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Flow Cytometry, Western Blot

    Enhancement of autophagy upregulates Sirt3 expression, and overexpression of Sirt3 reduces oxidative stress and rescues hematopoietic aging in autophagy‐defective mice. (a) Rapamycin upregulates the mammalian autophagy‐essential genes in wild‐type HSC‐enriched hematopoietic cells. Relative expression levels were measured by quantitative PCR. (b) Rapamycin increases the co‐localization between LC3 and Lamp1, an indicator for the formation of autolysosomes, in wild‐type HSC‐enriched hematopoietic cells. The representative images and statistical results were taken and analyzed by Amnis ImageStream image flow cytometer. Rapamycin 200 ng/ml, bafilomycin 10 nM. (c) Quantitative PCR detection of ex vivo autophagy‐induced transcription levels by rapamycin in primary HSC‐enriched hematopoietic cells of wild‐type and Atg7‐deleted mice for Sirt3 (left) and the rest of Sirtuin family members (right). (d) Ex vivo activation of autophagy by starvation selectively upregulates Sirt3 transcription in primary HSC‐enriched hematopoietic cells of wild‐type mice. Transcription levels for Sirtuin family members were detected by quantitative PCR. (e) In vivo activation of autophagy by progressive starvation upregulates Sirt3 transcription. Transcription levels for Sirt3 (left) and autophagy‐essential genes (right) in the HSC‐enriched hematopoietic cells from long‐term progressively starved mice were detected by quantitative PCR. Starvation was achieved by calorie restriction where the amount of feed for the mice was increasingly reduced by 10% of that fed last week, for a total of 4 weeks. (f‐j) Overexpression of Sirt3 reduces oxidative stress and rescues hematopoietic aging in autophagy‐defective mice. Diagrammatic sketch showing the generation of the mouse model with in vivo ectopic overexpression of Sirt3 by lentivirus infection in mouse HSC‐enriched hematopoietic cells, followed by the infected cell transplantation (f); Western blotting analysis of Sirt3 overexpression vector in mammalian cells infected by lentivirus and quantitative result for Sirt3 overexpression in NIH3T3 cells (g); flow cytometric examination of ex vivo ROS levels in the HSC‐enriched cells (h); donor hematopoietic engraft percentage measured 8 weeks after transplantation (i) and hematopoietic reconstitution for myeloid and lymphoid lineages (j) in the Atg7 +/+ and Atg7 −/− host mice infected by control or Sirt3 overexpression lentivirus

    Journal: Aging Cell

    Article Title: Autophagy‐Sirt3 axis decelerates hematopoietic aging

    doi: 10.1111/acel.13232

    Figure Lengend Snippet: Enhancement of autophagy upregulates Sirt3 expression, and overexpression of Sirt3 reduces oxidative stress and rescues hematopoietic aging in autophagy‐defective mice. (a) Rapamycin upregulates the mammalian autophagy‐essential genes in wild‐type HSC‐enriched hematopoietic cells. Relative expression levels were measured by quantitative PCR. (b) Rapamycin increases the co‐localization between LC3 and Lamp1, an indicator for the formation of autolysosomes, in wild‐type HSC‐enriched hematopoietic cells. The representative images and statistical results were taken and analyzed by Amnis ImageStream image flow cytometer. Rapamycin 200 ng/ml, bafilomycin 10 nM. (c) Quantitative PCR detection of ex vivo autophagy‐induced transcription levels by rapamycin in primary HSC‐enriched hematopoietic cells of wild‐type and Atg7‐deleted mice for Sirt3 (left) and the rest of Sirtuin family members (right). (d) Ex vivo activation of autophagy by starvation selectively upregulates Sirt3 transcription in primary HSC‐enriched hematopoietic cells of wild‐type mice. Transcription levels for Sirtuin family members were detected by quantitative PCR. (e) In vivo activation of autophagy by progressive starvation upregulates Sirt3 transcription. Transcription levels for Sirt3 (left) and autophagy‐essential genes (right) in the HSC‐enriched hematopoietic cells from long‐term progressively starved mice were detected by quantitative PCR. Starvation was achieved by calorie restriction where the amount of feed for the mice was increasingly reduced by 10% of that fed last week, for a total of 4 weeks. (f‐j) Overexpression of Sirt3 reduces oxidative stress and rescues hematopoietic aging in autophagy‐defective mice. Diagrammatic sketch showing the generation of the mouse model with in vivo ectopic overexpression of Sirt3 by lentivirus infection in mouse HSC‐enriched hematopoietic cells, followed by the infected cell transplantation (f); Western blotting analysis of Sirt3 overexpression vector in mammalian cells infected by lentivirus and quantitative result for Sirt3 overexpression in NIH3T3 cells (g); flow cytometric examination of ex vivo ROS levels in the HSC‐enriched cells (h); donor hematopoietic engraft percentage measured 8 weeks after transplantation (i) and hematopoietic reconstitution for myeloid and lymphoid lineages (j) in the Atg7 +/+ and Atg7 −/− host mice infected by control or Sirt3 overexpression lentivirus

    Article Snippet: Image flow cytometric analysis of co‐localization of CD34 (555821, BD Pharmingen), CD45 (563879, BD Pharmingen), LC3 (14079S, Cell Signaling Technology), and Lamp1 (ab24170, Abcam) was performed with Amnis ImageStream Image Flow Cytometer (Amnis, Merck Millipore).

    Techniques: Expressing, Over Expression, Real-time Polymerase Chain Reaction, Flow Cytometry, Ex Vivo, Activation Assay, In Vivo, Infection, Transplantation Assay, Western Blot, Plasmid Preparation