polyclonal antibodies against mouse mst1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc polyclonal antibodies against mouse mst1
    A Flow cytometry analysis (left) and mean fluorescence intensity (MFI, right) of <t>Mst1</t> levels in NKp (CD3 − CD122 + CD11b − NK1.1 − ), immature NK (CD3 − CD122 + CD11b − NK1.1 + , imNK) and mature NK (CD3 − CD122 + CD11b + NK1.1 + , mNK) cells from the spleen of wild-type mice (WT) ( n = 4). B Flow cytometry analysis (left) and MFI intensity (right) of Mst1 levels in CD27 SP (CD3 − NKp46 + CD27 + CD11b − ), DP (CD3 − NKp46 + CD27 + CD11b + ) and CD11b SP (CD3 − NKp46 + CD27 − CD11b + ) NK cells from the spleen of WT mice ( n = 4). C Flow cytometry analysis (left) and MFI intensity (right) of p-Mob1 expression in splenic NK cells before and after stimulation with 50 ng/mL IL-15 for 30 min ( n = 4). D – F Flow cytometry analysis of CD3 − NK1.1 + NK cells in bone marrow (BM) and spleen from WT and the indicated conditional knockout mice ( D ). The percentages ( E ) and the absolute numbers ( F ) of NK cells in spleen and BM from the indicated mice ( n = 5). The percentages of NKp, imNK, mNK cells on gated CD3 − CD122 + cells ( G ) and CD27 SP, DP, CD11b SP NK cell subsets on gated CD3 − NKp46 + cells ( H ) in BM (left) and spleen (right) from the indicated mice ( n = 5). I Percentages of CD127 + (left) and KLRG1 + (right) NK cells in the BM and spleen from the Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice ( n = 3). J Representative plots (left) and summary data (right) of the percentage of NK cells in the BM and spleen from BM chimera mice ( n = 5). Data of A – I are representative of three independent experiments with similar results. Data of J are representative of two independent experiments with similar results.
    Polyclonal Antibodies Against Mouse Mst1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polyclonal antibodies against mouse mst1/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    polyclonal antibodies against mouse mst1 - by Bioz Stars, 2024-06
    86/100 stars

    Images

    1) Product Images from "Hippo kinases Mst1 and Mst2 maintain NK cell homeostasis by orchestrating metabolic state and transcriptional activity"

    Article Title: Hippo kinases Mst1 and Mst2 maintain NK cell homeostasis by orchestrating metabolic state and transcriptional activity

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-024-06828-x

    A Flow cytometry analysis (left) and mean fluorescence intensity (MFI, right) of Mst1 levels in NKp (CD3 − CD122 + CD11b − NK1.1 − ), immature NK (CD3 − CD122 + CD11b − NK1.1 + , imNK) and mature NK (CD3 − CD122 + CD11b + NK1.1 + , mNK) cells from the spleen of wild-type mice (WT) ( n = 4). B Flow cytometry analysis (left) and MFI intensity (right) of Mst1 levels in CD27 SP (CD3 − NKp46 + CD27 + CD11b − ), DP (CD3 − NKp46 + CD27 + CD11b + ) and CD11b SP (CD3 − NKp46 + CD27 − CD11b + ) NK cells from the spleen of WT mice ( n = 4). C Flow cytometry analysis (left) and MFI intensity (right) of p-Mob1 expression in splenic NK cells before and after stimulation with 50 ng/mL IL-15 for 30 min ( n = 4). D – F Flow cytometry analysis of CD3 − NK1.1 + NK cells in bone marrow (BM) and spleen from WT and the indicated conditional knockout mice ( D ). The percentages ( E ) and the absolute numbers ( F ) of NK cells in spleen and BM from the indicated mice ( n = 5). The percentages of NKp, imNK, mNK cells on gated CD3 − CD122 + cells ( G ) and CD27 SP, DP, CD11b SP NK cell subsets on gated CD3 − NKp46 + cells ( H ) in BM (left) and spleen (right) from the indicated mice ( n = 5). I Percentages of CD127 + (left) and KLRG1 + (right) NK cells in the BM and spleen from the Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice ( n = 3). J Representative plots (left) and summary data (right) of the percentage of NK cells in the BM and spleen from BM chimera mice ( n = 5). Data of A – I are representative of three independent experiments with similar results. Data of J are representative of two independent experiments with similar results.
    Figure Legend Snippet: A Flow cytometry analysis (left) and mean fluorescence intensity (MFI, right) of Mst1 levels in NKp (CD3 − CD122 + CD11b − NK1.1 − ), immature NK (CD3 − CD122 + CD11b − NK1.1 + , imNK) and mature NK (CD3 − CD122 + CD11b + NK1.1 + , mNK) cells from the spleen of wild-type mice (WT) ( n = 4). B Flow cytometry analysis (left) and MFI intensity (right) of Mst1 levels in CD27 SP (CD3 − NKp46 + CD27 + CD11b − ), DP (CD3 − NKp46 + CD27 + CD11b + ) and CD11b SP (CD3 − NKp46 + CD27 − CD11b + ) NK cells from the spleen of WT mice ( n = 4). C Flow cytometry analysis (left) and MFI intensity (right) of p-Mob1 expression in splenic NK cells before and after stimulation with 50 ng/mL IL-15 for 30 min ( n = 4). D – F Flow cytometry analysis of CD3 − NK1.1 + NK cells in bone marrow (BM) and spleen from WT and the indicated conditional knockout mice ( D ). The percentages ( E ) and the absolute numbers ( F ) of NK cells in spleen and BM from the indicated mice ( n = 5). The percentages of NKp, imNK, mNK cells on gated CD3 − CD122 + cells ( G ) and CD27 SP, DP, CD11b SP NK cell subsets on gated CD3 − NKp46 + cells ( H ) in BM (left) and spleen (right) from the indicated mice ( n = 5). I Percentages of CD127 + (left) and KLRG1 + (right) NK cells in the BM and spleen from the Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice ( n = 3). J Representative plots (left) and summary data (right) of the percentage of NK cells in the BM and spleen from BM chimera mice ( n = 5). Data of A – I are representative of three independent experiments with similar results. Data of J are representative of two independent experiments with similar results.

    Techniques Used: Flow Cytometry, Fluorescence, Expressing, Knock-Out

    Expression levels of IFN-γ ( A ) and CD107a ( B ) in total NK cells from spleen of Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice after stimulation with anti-Ly49D antibody or YAC-1 cells ( n = 3). Expression levels of IFN-γ ( C ) and CD107a ( D ) in CD27 + CD11b + DP (left) and CD27 − CD11b + CD11b SP (right) subsets of NK cell from spleen of Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice after stimulation with anti-Ly49D antibody or YAC-1 cells ( n = 3). E Representative flow cytometry plots (left) and the percentage (right) of rejected β2m −/− splenocytes cells in the spleen and lymph nodes (LN) from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice ( n = 4). F Representative flow cytometry plots (left) and the percentage (right) of rejected RMA-S cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice ( n ≥ 4). G Representative images (left), the lung weights (middle) and the number of tumor nodules (right) from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice ( n ≥ 4). H FACS analysis of the cytotoxicity of NK cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice against RMA-S cells was detected by 7AAD staining at different E : T ratios ( n = 4). Data are representative of three independent experiments with similar results.
    Figure Legend Snippet: Expression levels of IFN-γ ( A ) and CD107a ( B ) in total NK cells from spleen of Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice after stimulation with anti-Ly49D antibody or YAC-1 cells ( n = 3). Expression levels of IFN-γ ( C ) and CD107a ( D ) in CD27 + CD11b + DP (left) and CD27 − CD11b + CD11b SP (right) subsets of NK cell from spleen of Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice after stimulation with anti-Ly49D antibody or YAC-1 cells ( n = 3). E Representative flow cytometry plots (left) and the percentage (right) of rejected β2m −/− splenocytes cells in the spleen and lymph nodes (LN) from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice ( n = 4). F Representative flow cytometry plots (left) and the percentage (right) of rejected RMA-S cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice ( n ≥ 4). G Representative images (left), the lung weights (middle) and the number of tumor nodules (right) from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice ( n ≥ 4). H FACS analysis of the cytotoxicity of NK cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice against RMA-S cells was detected by 7AAD staining at different E : T ratios ( n = 4). Data are representative of three independent experiments with similar results.

    Techniques Used: Expressing, Flow Cytometry, Staining

    A – F RNA sequencing was performed on NK cells sorted from splenocytes in Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice. A The volcano plot illustrates genes that exhibit differential expression between NK cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice, with lines indicating a twofold difference (edgeR, p < 0.05). B The heatmap displays the expression levels of up- and downregulated genes in Mst1/2-depleted NK cells compared to control NK cells. Gene-set enrichment analysis of RNA-seq data reveals an enrichment of the related genes for cell cycle ( C ), G1 to cell cycle control ( D ) and cell population proliferation ( E ) in Mst1/2-deficient NK cells compared to WT NK cells. F Heatmap analysis of RNA-seq data demonstrates differentially expressed cell proliferation-related genes between NK cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice. G Representative plots (left) and statistic data (right) illustrate Ki-67 + NK cell subsets in spleen from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice ( n ≥ 4). Representative plots (left) and statistic data (right) illustrate Annexin V + ( H ) and active caspase3 + ( I ) NK cells in spleen from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice ( n ≥ 4). Flow cytometry analysis (left) and MFI intensity (right) show Bim ( J ) and Bcl-2 ( K ) levels in splenic NK cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice ( n = 5). Data of G – K are representative of three independent experiments with similar results.
    Figure Legend Snippet: A – F RNA sequencing was performed on NK cells sorted from splenocytes in Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice. A The volcano plot illustrates genes that exhibit differential expression between NK cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice, with lines indicating a twofold difference (edgeR, p < 0.05). B The heatmap displays the expression levels of up- and downregulated genes in Mst1/2-depleted NK cells compared to control NK cells. Gene-set enrichment analysis of RNA-seq data reveals an enrichment of the related genes for cell cycle ( C ), G1 to cell cycle control ( D ) and cell population proliferation ( E ) in Mst1/2-deficient NK cells compared to WT NK cells. F Heatmap analysis of RNA-seq data demonstrates differentially expressed cell proliferation-related genes between NK cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice. G Representative plots (left) and statistic data (right) illustrate Ki-67 + NK cell subsets in spleen from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice ( n ≥ 4). Representative plots (left) and statistic data (right) illustrate Annexin V + ( H ) and active caspase3 + ( I ) NK cells in spleen from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice ( n ≥ 4). Flow cytometry analysis (left) and MFI intensity (right) show Bim ( J ) and Bcl-2 ( K ) levels in splenic NK cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice ( n = 5). Data of G – K are representative of three independent experiments with similar results.

    Techniques Used: RNA Sequencing Assay, Expressing, Control, Flow Cytometry

    Seahorse extracellular flux analysis was performed to measure the oxygen consumption rate (OCR) in NK cells from the BM ( A ) and spleen ( B ) of Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice. Flow cytometry analysis (left) and MFI intensity (right) illustrate the expression levels of mitoTracker ( C , D ), TMRM ( E , F ), total cellular ROS ( G , H ), mitochondrial ROS ( I, J ) and p-S6 ( K , L ) in NK cells from BM ( C , E , G , I , K ) and spleen ( D , F , H , J , L ) of Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice ( n ≥ 4). M Representative flow cytometry plot (left) and summary data (right) display the percentage of dead cells in spleen from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice fed water with or without NAC (1.5 g/L) for 30 days starting at 21-day-old ( n ≥ 4). N Representative flow cytometry plot (left) and summary data (right) show the number of CD3 − CD122 + cells in spleen from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice fed water with or without NAC (1.5 g/L) for 30 days starting at 21-day-old ( n ≥ 4). Data of C – L are representative of three independent experiments with similar results. Data of M and N are representative of two independent experiments with similar results.
    Figure Legend Snippet: Seahorse extracellular flux analysis was performed to measure the oxygen consumption rate (OCR) in NK cells from the BM ( A ) and spleen ( B ) of Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice. Flow cytometry analysis (left) and MFI intensity (right) illustrate the expression levels of mitoTracker ( C , D ), TMRM ( E , F ), total cellular ROS ( G , H ), mitochondrial ROS ( I, J ) and p-S6 ( K , L ) in NK cells from BM ( C , E , G , I , K ) and spleen ( D , F , H , J , L ) of Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice ( n ≥ 4). M Representative flow cytometry plot (left) and summary data (right) display the percentage of dead cells in spleen from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice fed water with or without NAC (1.5 g/L) for 30 days starting at 21-day-old ( n ≥ 4). N Representative flow cytometry plot (left) and summary data (right) show the number of CD3 − CD122 + cells in spleen from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice fed water with or without NAC (1.5 g/L) for 30 days starting at 21-day-old ( n ≥ 4). Data of C – L are representative of three independent experiments with similar results. Data of M and N are representative of two independent experiments with similar results.

    Techniques Used: Flow Cytometry, Expressing

    A Heatmap analysis of RNA-seq data reveals differential expression of certain transcription factors between NK cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice. Flow cytometry analysis ( B ) and MFI intensity ( C ) demonstrate the levels of Eomes, T-bet, E4BP4 and TCF1 in splenic NK cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice ( n ≥ 4). D Gene-set enrichment analysis of RNA-seq data indicates a negative enrichment of TCF1-activated genes in Mst1/2-deficient NK cells compared to WT NK cells. E Flow cytometry analysis (left) and MFI intensity (right) reveal the levels of TCF1 in NK cells from the spleen of Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice upon stimulation with or without IL-15 (10 ng/mL) for 16 h ( n = 4). F – I Representative plots (left panel) and summary data (right panel) illustrate the percentage of NK cells ( F ), the MFI of total cellular ROS levels ( G ), the percentage of 7AAD + NK cells (H) and distribution pattern of different NK cell subsets (I) in the spleen of BM chimeric mice ( n ≥ 3). Data B , C and E are representative of three independent experiments with similar results. Data F – I are representative of two independent experiments with similar results.
    Figure Legend Snippet: A Heatmap analysis of RNA-seq data reveals differential expression of certain transcription factors between NK cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice. Flow cytometry analysis ( B ) and MFI intensity ( C ) demonstrate the levels of Eomes, T-bet, E4BP4 and TCF1 in splenic NK cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice ( n ≥ 4). D Gene-set enrichment analysis of RNA-seq data indicates a negative enrichment of TCF1-activated genes in Mst1/2-deficient NK cells compared to WT NK cells. E Flow cytometry analysis (left) and MFI intensity (right) reveal the levels of TCF1 in NK cells from the spleen of Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice upon stimulation with or without IL-15 (10 ng/mL) for 16 h ( n = 4). F – I Representative plots (left panel) and summary data (right panel) illustrate the percentage of NK cells ( F ), the MFI of total cellular ROS levels ( G ), the percentage of 7AAD + NK cells (H) and distribution pattern of different NK cell subsets (I) in the spleen of BM chimeric mice ( n ≥ 3). Data B , C and E are representative of three independent experiments with similar results. Data F – I are representative of two independent experiments with similar results.

    Techniques Used: RNA Sequencing Assay, Expressing, Flow Cytometry

    A , B Flow cytometry analysis (left) and MFI intensity (right) demonstrate the expression levels of p-STAT5, p-S6, p-AKT473 and p-Erk in NK cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice upon stimulation with or without IL-15 (50 ng/mL) for 30 min ( n ≥ 4). C Gene-set enrichment analysis of RNA-seq data shows the enrichment of hallmark-IL6-JAK-STAT3-signaling related genes in Mst1/2-deficient NK cells compared to WT NK cells. D Flow cytometry analysis (left) and MFI intensity (right) reveals the expression level of p-STAT3 (Ser727) in NK cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice upon stimulation with or without IL-15 (50 ng/mL) for 30 min ( n = 4). E ChIP-qPCR analysis demonstrated binding of p-STAT3 conserved motifs in the Tcf7 5′ regulatory region (−17.5 kb), while no binding was observed in a region without p-STAT3-binding motifs (+0.2 kb), serving as a negative control. The experiments were using sorted NK cells from WT mice with anti-p-STAT3 antibody or isotype-matched IgG. F – I WT splenocytes were treated with DMSO, Mst1 inhibitor XMU-MP-1 (1 mM, MedChemExpress) or p-STAT3 inhibitor Stattic (1 mM, MedChemExpress) for 24 h, followed by flow cytometry analysis. The representative plots (left) and summary data (right) show the expression level of p-STAT3 (F), TCF1 ( G ), total cellular ROS ( H ) and the percentage of 7AAD + NK cells ( I ) ( n = 4). J Schematic illustration of Mst1/2 regulate NK cell survival and function via controlling metabolic state and transcriptional activity. Data A , B and D – I are representative of three independent experiments with similar results.
    Figure Legend Snippet: A , B Flow cytometry analysis (left) and MFI intensity (right) demonstrate the expression levels of p-STAT5, p-S6, p-AKT473 and p-Erk in NK cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice upon stimulation with or without IL-15 (50 ng/mL) for 30 min ( n ≥ 4). C Gene-set enrichment analysis of RNA-seq data shows the enrichment of hallmark-IL6-JAK-STAT3-signaling related genes in Mst1/2-deficient NK cells compared to WT NK cells. D Flow cytometry analysis (left) and MFI intensity (right) reveals the expression level of p-STAT3 (Ser727) in NK cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice upon stimulation with or without IL-15 (50 ng/mL) for 30 min ( n = 4). E ChIP-qPCR analysis demonstrated binding of p-STAT3 conserved motifs in the Tcf7 5′ regulatory region (−17.5 kb), while no binding was observed in a region without p-STAT3-binding motifs (+0.2 kb), serving as a negative control. The experiments were using sorted NK cells from WT mice with anti-p-STAT3 antibody or isotype-matched IgG. F – I WT splenocytes were treated with DMSO, Mst1 inhibitor XMU-MP-1 (1 mM, MedChemExpress) or p-STAT3 inhibitor Stattic (1 mM, MedChemExpress) for 24 h, followed by flow cytometry analysis. The representative plots (left) and summary data (right) show the expression level of p-STAT3 (F), TCF1 ( G ), total cellular ROS ( H ) and the percentage of 7AAD + NK cells ( I ) ( n = 4). J Schematic illustration of Mst1/2 regulate NK cell survival and function via controlling metabolic state and transcriptional activity. Data A , B and D – I are representative of three independent experiments with similar results.

    Techniques Used: Flow Cytometry, Expressing, RNA Sequencing Assay, Binding Assay, Negative Control, Activity Assay

    rabbit polyclonal antibody against mouse spink1  (Cell Signaling Technology Inc)


    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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    Cell Signaling Technology Inc rabbit polyclonal antibody against mouse spink1
    Rabbit Polyclonal Antibody Against Mouse Spink1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 1 article reviews
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    mouse anti rat pten polyclonal antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc mouse anti rat pten polyclonal antibody
    Mouse Anti Rat Pten Polyclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mouse anti β actin polyclonal antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc mouse anti β actin polyclonal antibody
    Mouse Anti β Actin Polyclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti β actin polyclonal antibody/product/Cell Signaling Technology Inc
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    mouse anti β actin polyclonal antibody  (Cell Signaling Technology Inc)


    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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    Cell Signaling Technology Inc mouse anti β actin polyclonal antibody
    Mouse Anti β Actin Polyclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti β actin polyclonal antibody/product/Cell Signaling Technology Inc
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    rabbit polyclonal anti mouse irg1 acod1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit polyclonal anti mouse irg1 acod1
    (A) Schematic of ITA generation by cis -aconitate decarboxylase <t>(Acod1).</t> (B and C) PRs or PRp cells were grown in the indicated conditions for 24 h. (B) Lysates were collected and immunoblotted as noted. (C) Gene expression of <t>Acod1</t> by quantitative real-time PCR calculated as FC relative to Att PRs. (D) PRp cells were grown in the indicated conditions in the presence of DMSO or carbonyl cyanide m -chlorophenyl hydrazone (CCCP) (20 μM) for 48 h. Lysates were collected and immunoblotted as noted. (E) PRs or PRp cells were grown in the indicated conditions in the presence of DMSO or the nuclear factor κB (NF-κB) inhibitor BAY1170-82 (2.5 μM) for 9 h. Lysates were collected and immunoblotted as noted. Graphs represent data collected from a minimum of 3 biological replicates, and all western blotting experiments were independently repeated a minimum of three times with similar results. p values are calculated by one-way ANOVA followed by Tukey’s test in (C). Data are mean ± SEM.
    Rabbit Polyclonal Anti Mouse Irg1 Acod1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit polyclonal anti mouse irg1 acod1/product/Cell Signaling Technology Inc
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    1) Product Images from "Acod1 expression in cancer cells promotes immune evasion through the generation of inhibitory peptides"

    Article Title: Acod1 expression in cancer cells promotes immune evasion through the generation of inhibitory peptides

    Journal: Cell reports

    doi: 10.1016/j.celrep.2024.113984

    (A) Schematic of ITA generation by cis -aconitate decarboxylase (Acod1). (B and C) PRs or PRp cells were grown in the indicated conditions for 24 h. (B) Lysates were collected and immunoblotted as noted. (C) Gene expression of Acod1 by quantitative real-time PCR calculated as FC relative to Att PRs. (D) PRp cells were grown in the indicated conditions in the presence of DMSO or carbonyl cyanide m -chlorophenyl hydrazone (CCCP) (20 μM) for 48 h. Lysates were collected and immunoblotted as noted. (E) PRs or PRp cells were grown in the indicated conditions in the presence of DMSO or the nuclear factor κB (NF-κB) inhibitor BAY1170-82 (2.5 μM) for 9 h. Lysates were collected and immunoblotted as noted. Graphs represent data collected from a minimum of 3 biological replicates, and all western blotting experiments were independently repeated a minimum of three times with similar results. p values are calculated by one-way ANOVA followed by Tukey’s test in (C). Data are mean ± SEM.
    Figure Legend Snippet: (A) Schematic of ITA generation by cis -aconitate decarboxylase (Acod1). (B and C) PRs or PRp cells were grown in the indicated conditions for 24 h. (B) Lysates were collected and immunoblotted as noted. (C) Gene expression of Acod1 by quantitative real-time PCR calculated as FC relative to Att PRs. (D) PRp cells were grown in the indicated conditions in the presence of DMSO or carbonyl cyanide m -chlorophenyl hydrazone (CCCP) (20 μM) for 48 h. Lysates were collected and immunoblotted as noted. (E) PRs or PRp cells were grown in the indicated conditions in the presence of DMSO or the nuclear factor κB (NF-κB) inhibitor BAY1170-82 (2.5 μM) for 9 h. Lysates were collected and immunoblotted as noted. Graphs represent data collected from a minimum of 3 biological replicates, and all western blotting experiments were independently repeated a minimum of three times with similar results. p values are calculated by one-way ANOVA followed by Tukey’s test in (C). Data are mean ± SEM.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot

    (A) Experimental design of in vivo experiment. (B) Western blot for Acod1 of cells injected for tumor experiments. (C) PRp Scr or PRp Acod1 KO cells were subcutaneously injected in mice and received either isotype control (IgG) or αPD-1 monoclonal antibody (mAb) when tumors reached ≥50 mm 3 . Data are mean ± SEM (n = 6–10 per group), and p values were calculated by two-way ANOVA. (D and E) Percentage of CD4 + (D) or CD8 + (E) T cells within each tumor. Data represent the mean ± SEM (n = 4 mice/group), and p values were calculated by two-tailed Student’s t test. n.s., not significant. (F) Representative images of immunofluorescence (IF) staining of GzmB + (red) and TUNEL + (green) cells in tumors from the indicated conditions described in (A). Scale bars of low and high magnification represent 200 and 50 μm, respectively. DAPI, 4′,6-diamidino-2-phenylindole. (G) Quantification of relative GzmB intensity (top) or average number of TUNEL + cells (bottom) with SD from six random fields of view (FOVs). The p values were calculated by one-way ANOVA. n.s., not significant.
    Figure Legend Snippet: (A) Experimental design of in vivo experiment. (B) Western blot for Acod1 of cells injected for tumor experiments. (C) PRp Scr or PRp Acod1 KO cells were subcutaneously injected in mice and received either isotype control (IgG) or αPD-1 monoclonal antibody (mAb) when tumors reached ≥50 mm 3 . Data are mean ± SEM (n = 6–10 per group), and p values were calculated by two-way ANOVA. (D and E) Percentage of CD4 + (D) or CD8 + (E) T cells within each tumor. Data represent the mean ± SEM (n = 4 mice/group), and p values were calculated by two-tailed Student’s t test. n.s., not significant. (F) Representative images of immunofluorescence (IF) staining of GzmB + (red) and TUNEL + (green) cells in tumors from the indicated conditions described in (A). Scale bars of low and high magnification represent 200 and 50 μm, respectively. DAPI, 4′,6-diamidino-2-phenylindole. (G) Quantification of relative GzmB intensity (top) or average number of TUNEL + cells (bottom) with SD from six random fields of view (FOVs). The p values were calculated by one-way ANOVA. n.s., not significant.

    Techniques Used: In Vivo, Western Blot, Injection, Two Tailed Test, Immunofluorescence, Staining, TUNEL Assay

    (A) Percentage of EdU + CD8 + T cells following 48 h of activation with αCD3/CD28 in indicated conditioned medium (CM). (B) Representative histograms of violet proliferation dye 450 (VPD450) dilution in CD8 + T cells following 72 h of activation with αCD3/CD28 in indicated CM. (C) Histograms of CD44 (left) and CD25 (right) expression in CD8 + T cells following 72 h of activation with αCD3/CD28 in indicated CM (n = 3). (D) Percentage of CD44 + (left) and CD25 + (right) CD8 + T cells following 72 h of activation with αCD3/CD28 in indicated CM. (E) Cells grown for 24 h in Det conditions. Lysates were collected and immunoblotted as noted. (F) Percentage of EdU + CD8 + T cells as described in (A). (G) Histograms of CD44 (left) and CD25 (right) expression as described in (C). Scr, PRp Scr CM; KO, PRp Acod1 KO CM (n = 3). (H) Percentage of CD44 + (left) and CD25 + (right) CD8 + T cells as described in (D). Data from EdU experiments (A and F) represent the means ± SEM of triplicate wells, and p values were calculated by one-way ANOVA analysis. Data are representative of three independent experiments. The p values for the T cell activation experiments (D and H) were calculated using a paired, two-tailed t test (n = 4 mice/group). n.s., not significant. Western blotting and VPD450 dilution experiments were independently repeated a minimum of three times with similar results.
    Figure Legend Snippet: (A) Percentage of EdU + CD8 + T cells following 48 h of activation with αCD3/CD28 in indicated conditioned medium (CM). (B) Representative histograms of violet proliferation dye 450 (VPD450) dilution in CD8 + T cells following 72 h of activation with αCD3/CD28 in indicated CM. (C) Histograms of CD44 (left) and CD25 (right) expression in CD8 + T cells following 72 h of activation with αCD3/CD28 in indicated CM (n = 3). (D) Percentage of CD44 + (left) and CD25 + (right) CD8 + T cells following 72 h of activation with αCD3/CD28 in indicated CM. (E) Cells grown for 24 h in Det conditions. Lysates were collected and immunoblotted as noted. (F) Percentage of EdU + CD8 + T cells as described in (A). (G) Histograms of CD44 (left) and CD25 (right) expression as described in (C). Scr, PRp Scr CM; KO, PRp Acod1 KO CM (n = 3). (H) Percentage of CD44 + (left) and CD25 + (right) CD8 + T cells as described in (D). Data from EdU experiments (A and F) represent the means ± SEM of triplicate wells, and p values were calculated by one-way ANOVA analysis. Data are representative of three independent experiments. The p values for the T cell activation experiments (D and H) were calculated using a paired, two-tailed t test (n = 4 mice/group). n.s., not significant. Western blotting and VPD450 dilution experiments were independently repeated a minimum of three times with similar results.

    Techniques Used: Activation Assay, Expressing, Two Tailed Test, Western Blot

    KEY RESOURCES TABLE
    Figure Legend Snippet: KEY RESOURCES TABLE

    Techniques Used: Recombinant, Lysis, Staining, Transfection, In Situ, Cell Isolation, Plasmid Preparation, Software

    polyclonal anti mouse igg peroxidase  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit primary polyclonal antibodies against mouse pten
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    Image Search Results


    A Flow cytometry analysis (left) and mean fluorescence intensity (MFI, right) of Mst1 levels in NKp (CD3 − CD122 + CD11b − NK1.1 − ), immature NK (CD3 − CD122 + CD11b − NK1.1 + , imNK) and mature NK (CD3 − CD122 + CD11b + NK1.1 + , mNK) cells from the spleen of wild-type mice (WT) ( n = 4). B Flow cytometry analysis (left) and MFI intensity (right) of Mst1 levels in CD27 SP (CD3 − NKp46 + CD27 + CD11b − ), DP (CD3 − NKp46 + CD27 + CD11b + ) and CD11b SP (CD3 − NKp46 + CD27 − CD11b + ) NK cells from the spleen of WT mice ( n = 4). C Flow cytometry analysis (left) and MFI intensity (right) of p-Mob1 expression in splenic NK cells before and after stimulation with 50 ng/mL IL-15 for 30 min ( n = 4). D – F Flow cytometry analysis of CD3 − NK1.1 + NK cells in bone marrow (BM) and spleen from WT and the indicated conditional knockout mice ( D ). The percentages ( E ) and the absolute numbers ( F ) of NK cells in spleen and BM from the indicated mice ( n = 5). The percentages of NKp, imNK, mNK cells on gated CD3 − CD122 + cells ( G ) and CD27 SP, DP, CD11b SP NK cell subsets on gated CD3 − NKp46 + cells ( H ) in BM (left) and spleen (right) from the indicated mice ( n = 5). I Percentages of CD127 + (left) and KLRG1 + (right) NK cells in the BM and spleen from the Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice ( n = 3). J Representative plots (left) and summary data (right) of the percentage of NK cells in the BM and spleen from BM chimera mice ( n = 5). Data of A – I are representative of three independent experiments with similar results. Data of J are representative of two independent experiments with similar results.

    Journal: Cell Death & Disease

    Article Title: Hippo kinases Mst1 and Mst2 maintain NK cell homeostasis by orchestrating metabolic state and transcriptional activity

    doi: 10.1038/s41419-024-06828-x

    Figure Lengend Snippet: A Flow cytometry analysis (left) and mean fluorescence intensity (MFI, right) of Mst1 levels in NKp (CD3 − CD122 + CD11b − NK1.1 − ), immature NK (CD3 − CD122 + CD11b − NK1.1 + , imNK) and mature NK (CD3 − CD122 + CD11b + NK1.1 + , mNK) cells from the spleen of wild-type mice (WT) ( n = 4). B Flow cytometry analysis (left) and MFI intensity (right) of Mst1 levels in CD27 SP (CD3 − NKp46 + CD27 + CD11b − ), DP (CD3 − NKp46 + CD27 + CD11b + ) and CD11b SP (CD3 − NKp46 + CD27 − CD11b + ) NK cells from the spleen of WT mice ( n = 4). C Flow cytometry analysis (left) and MFI intensity (right) of p-Mob1 expression in splenic NK cells before and after stimulation with 50 ng/mL IL-15 for 30 min ( n = 4). D – F Flow cytometry analysis of CD3 − NK1.1 + NK cells in bone marrow (BM) and spleen from WT and the indicated conditional knockout mice ( D ). The percentages ( E ) and the absolute numbers ( F ) of NK cells in spleen and BM from the indicated mice ( n = 5). The percentages of NKp, imNK, mNK cells on gated CD3 − CD122 + cells ( G ) and CD27 SP, DP, CD11b SP NK cell subsets on gated CD3 − NKp46 + cells ( H ) in BM (left) and spleen (right) from the indicated mice ( n = 5). I Percentages of CD127 + (left) and KLRG1 + (right) NK cells in the BM and spleen from the Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice ( n = 3). J Representative plots (left) and summary data (right) of the percentage of NK cells in the BM and spleen from BM chimera mice ( n = 5). Data of A – I are representative of three independent experiments with similar results. Data of J are representative of two independent experiments with similar results.

    Article Snippet: Monoclonal antibodies against mouse TCF1 (Cat#9066S), phospho-Mob1 (Thr35) (Cat#8699), Bim (C34C5) Rabbit mAb (Cat#12186), Phospho-Stat5 (Tyr694) (Cat#9359), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cat#4370S) and polyclonal antibodies against mouse Mst1 (Cat#3682) were obtained from Cell Signaling Technology (Beverly, MA).

    Techniques: Flow Cytometry, Fluorescence, Expressing, Knock-Out

    Expression levels of IFN-γ ( A ) and CD107a ( B ) in total NK cells from spleen of Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice after stimulation with anti-Ly49D antibody or YAC-1 cells ( n = 3). Expression levels of IFN-γ ( C ) and CD107a ( D ) in CD27 + CD11b + DP (left) and CD27 − CD11b + CD11b SP (right) subsets of NK cell from spleen of Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice after stimulation with anti-Ly49D antibody or YAC-1 cells ( n = 3). E Representative flow cytometry plots (left) and the percentage (right) of rejected β2m −/− splenocytes cells in the spleen and lymph nodes (LN) from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice ( n = 4). F Representative flow cytometry plots (left) and the percentage (right) of rejected RMA-S cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice ( n ≥ 4). G Representative images (left), the lung weights (middle) and the number of tumor nodules (right) from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice ( n ≥ 4). H FACS analysis of the cytotoxicity of NK cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice against RMA-S cells was detected by 7AAD staining at different E : T ratios ( n = 4). Data are representative of three independent experiments with similar results.

    Journal: Cell Death & Disease

    Article Title: Hippo kinases Mst1 and Mst2 maintain NK cell homeostasis by orchestrating metabolic state and transcriptional activity

    doi: 10.1038/s41419-024-06828-x

    Figure Lengend Snippet: Expression levels of IFN-γ ( A ) and CD107a ( B ) in total NK cells from spleen of Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice after stimulation with anti-Ly49D antibody or YAC-1 cells ( n = 3). Expression levels of IFN-γ ( C ) and CD107a ( D ) in CD27 + CD11b + DP (left) and CD27 − CD11b + CD11b SP (right) subsets of NK cell from spleen of Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice after stimulation with anti-Ly49D antibody or YAC-1 cells ( n = 3). E Representative flow cytometry plots (left) and the percentage (right) of rejected β2m −/− splenocytes cells in the spleen and lymph nodes (LN) from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice ( n = 4). F Representative flow cytometry plots (left) and the percentage (right) of rejected RMA-S cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice ( n ≥ 4). G Representative images (left), the lung weights (middle) and the number of tumor nodules (right) from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice ( n ≥ 4). H FACS analysis of the cytotoxicity of NK cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice against RMA-S cells was detected by 7AAD staining at different E : T ratios ( n = 4). Data are representative of three independent experiments with similar results.

    Article Snippet: Monoclonal antibodies against mouse TCF1 (Cat#9066S), phospho-Mob1 (Thr35) (Cat#8699), Bim (C34C5) Rabbit mAb (Cat#12186), Phospho-Stat5 (Tyr694) (Cat#9359), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cat#4370S) and polyclonal antibodies against mouse Mst1 (Cat#3682) were obtained from Cell Signaling Technology (Beverly, MA).

    Techniques: Expressing, Flow Cytometry, Staining

    A – F RNA sequencing was performed on NK cells sorted from splenocytes in Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice. A The volcano plot illustrates genes that exhibit differential expression between NK cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice, with lines indicating a twofold difference (edgeR, p < 0.05). B The heatmap displays the expression levels of up- and downregulated genes in Mst1/2-depleted NK cells compared to control NK cells. Gene-set enrichment analysis of RNA-seq data reveals an enrichment of the related genes for cell cycle ( C ), G1 to cell cycle control ( D ) and cell population proliferation ( E ) in Mst1/2-deficient NK cells compared to WT NK cells. F Heatmap analysis of RNA-seq data demonstrates differentially expressed cell proliferation-related genes between NK cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice. G Representative plots (left) and statistic data (right) illustrate Ki-67 + NK cell subsets in spleen from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice ( n ≥ 4). Representative plots (left) and statistic data (right) illustrate Annexin V + ( H ) and active caspase3 + ( I ) NK cells in spleen from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice ( n ≥ 4). Flow cytometry analysis (left) and MFI intensity (right) show Bim ( J ) and Bcl-2 ( K ) levels in splenic NK cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice ( n = 5). Data of G – K are representative of three independent experiments with similar results.

    Journal: Cell Death & Disease

    Article Title: Hippo kinases Mst1 and Mst2 maintain NK cell homeostasis by orchestrating metabolic state and transcriptional activity

    doi: 10.1038/s41419-024-06828-x

    Figure Lengend Snippet: A – F RNA sequencing was performed on NK cells sorted from splenocytes in Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice. A The volcano plot illustrates genes that exhibit differential expression between NK cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice, with lines indicating a twofold difference (edgeR, p < 0.05). B The heatmap displays the expression levels of up- and downregulated genes in Mst1/2-depleted NK cells compared to control NK cells. Gene-set enrichment analysis of RNA-seq data reveals an enrichment of the related genes for cell cycle ( C ), G1 to cell cycle control ( D ) and cell population proliferation ( E ) in Mst1/2-deficient NK cells compared to WT NK cells. F Heatmap analysis of RNA-seq data demonstrates differentially expressed cell proliferation-related genes between NK cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice. G Representative plots (left) and statistic data (right) illustrate Ki-67 + NK cell subsets in spleen from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice ( n ≥ 4). Representative plots (left) and statistic data (right) illustrate Annexin V + ( H ) and active caspase3 + ( I ) NK cells in spleen from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice ( n ≥ 4). Flow cytometry analysis (left) and MFI intensity (right) show Bim ( J ) and Bcl-2 ( K ) levels in splenic NK cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice ( n = 5). Data of G – K are representative of three independent experiments with similar results.

    Article Snippet: Monoclonal antibodies against mouse TCF1 (Cat#9066S), phospho-Mob1 (Thr35) (Cat#8699), Bim (C34C5) Rabbit mAb (Cat#12186), Phospho-Stat5 (Tyr694) (Cat#9359), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cat#4370S) and polyclonal antibodies against mouse Mst1 (Cat#3682) were obtained from Cell Signaling Technology (Beverly, MA).

    Techniques: RNA Sequencing Assay, Expressing, Control, Flow Cytometry

    Seahorse extracellular flux analysis was performed to measure the oxygen consumption rate (OCR) in NK cells from the BM ( A ) and spleen ( B ) of Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice. Flow cytometry analysis (left) and MFI intensity (right) illustrate the expression levels of mitoTracker ( C , D ), TMRM ( E , F ), total cellular ROS ( G , H ), mitochondrial ROS ( I, J ) and p-S6 ( K , L ) in NK cells from BM ( C , E , G , I , K ) and spleen ( D , F , H , J , L ) of Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice ( n ≥ 4). M Representative flow cytometry plot (left) and summary data (right) display the percentage of dead cells in spleen from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice fed water with or without NAC (1.5 g/L) for 30 days starting at 21-day-old ( n ≥ 4). N Representative flow cytometry plot (left) and summary data (right) show the number of CD3 − CD122 + cells in spleen from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice fed water with or without NAC (1.5 g/L) for 30 days starting at 21-day-old ( n ≥ 4). Data of C – L are representative of three independent experiments with similar results. Data of M and N are representative of two independent experiments with similar results.

    Journal: Cell Death & Disease

    Article Title: Hippo kinases Mst1 and Mst2 maintain NK cell homeostasis by orchestrating metabolic state and transcriptional activity

    doi: 10.1038/s41419-024-06828-x

    Figure Lengend Snippet: Seahorse extracellular flux analysis was performed to measure the oxygen consumption rate (OCR) in NK cells from the BM ( A ) and spleen ( B ) of Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice. Flow cytometry analysis (left) and MFI intensity (right) illustrate the expression levels of mitoTracker ( C , D ), TMRM ( E , F ), total cellular ROS ( G , H ), mitochondrial ROS ( I, J ) and p-S6 ( K , L ) in NK cells from BM ( C , E , G , I , K ) and spleen ( D , F , H , J , L ) of Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice ( n ≥ 4). M Representative flow cytometry plot (left) and summary data (right) display the percentage of dead cells in spleen from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice fed water with or without NAC (1.5 g/L) for 30 days starting at 21-day-old ( n ≥ 4). N Representative flow cytometry plot (left) and summary data (right) show the number of CD3 − CD122 + cells in spleen from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice fed water with or without NAC (1.5 g/L) for 30 days starting at 21-day-old ( n ≥ 4). Data of C – L are representative of three independent experiments with similar results. Data of M and N are representative of two independent experiments with similar results.

    Article Snippet: Monoclonal antibodies against mouse TCF1 (Cat#9066S), phospho-Mob1 (Thr35) (Cat#8699), Bim (C34C5) Rabbit mAb (Cat#12186), Phospho-Stat5 (Tyr694) (Cat#9359), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cat#4370S) and polyclonal antibodies against mouse Mst1 (Cat#3682) were obtained from Cell Signaling Technology (Beverly, MA).

    Techniques: Flow Cytometry, Expressing

    A Heatmap analysis of RNA-seq data reveals differential expression of certain transcription factors between NK cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice. Flow cytometry analysis ( B ) and MFI intensity ( C ) demonstrate the levels of Eomes, T-bet, E4BP4 and TCF1 in splenic NK cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice ( n ≥ 4). D Gene-set enrichment analysis of RNA-seq data indicates a negative enrichment of TCF1-activated genes in Mst1/2-deficient NK cells compared to WT NK cells. E Flow cytometry analysis (left) and MFI intensity (right) reveal the levels of TCF1 in NK cells from the spleen of Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice upon stimulation with or without IL-15 (10 ng/mL) for 16 h ( n = 4). F – I Representative plots (left panel) and summary data (right panel) illustrate the percentage of NK cells ( F ), the MFI of total cellular ROS levels ( G ), the percentage of 7AAD + NK cells (H) and distribution pattern of different NK cell subsets (I) in the spleen of BM chimeric mice ( n ≥ 3). Data B , C and E are representative of three independent experiments with similar results. Data F – I are representative of two independent experiments with similar results.

    Journal: Cell Death & Disease

    Article Title: Hippo kinases Mst1 and Mst2 maintain NK cell homeostasis by orchestrating metabolic state and transcriptional activity

    doi: 10.1038/s41419-024-06828-x

    Figure Lengend Snippet: A Heatmap analysis of RNA-seq data reveals differential expression of certain transcription factors between NK cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice. Flow cytometry analysis ( B ) and MFI intensity ( C ) demonstrate the levels of Eomes, T-bet, E4BP4 and TCF1 in splenic NK cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice ( n ≥ 4). D Gene-set enrichment analysis of RNA-seq data indicates a negative enrichment of TCF1-activated genes in Mst1/2-deficient NK cells compared to WT NK cells. E Flow cytometry analysis (left) and MFI intensity (right) reveal the levels of TCF1 in NK cells from the spleen of Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice upon stimulation with or without IL-15 (10 ng/mL) for 16 h ( n = 4). F – I Representative plots (left panel) and summary data (right panel) illustrate the percentage of NK cells ( F ), the MFI of total cellular ROS levels ( G ), the percentage of 7AAD + NK cells (H) and distribution pattern of different NK cell subsets (I) in the spleen of BM chimeric mice ( n ≥ 3). Data B , C and E are representative of three independent experiments with similar results. Data F – I are representative of two independent experiments with similar results.

    Article Snippet: Monoclonal antibodies against mouse TCF1 (Cat#9066S), phospho-Mob1 (Thr35) (Cat#8699), Bim (C34C5) Rabbit mAb (Cat#12186), Phospho-Stat5 (Tyr694) (Cat#9359), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cat#4370S) and polyclonal antibodies against mouse Mst1 (Cat#3682) were obtained from Cell Signaling Technology (Beverly, MA).

    Techniques: RNA Sequencing Assay, Expressing, Flow Cytometry

    A , B Flow cytometry analysis (left) and MFI intensity (right) demonstrate the expression levels of p-STAT5, p-S6, p-AKT473 and p-Erk in NK cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice upon stimulation with or without IL-15 (50 ng/mL) for 30 min ( n ≥ 4). C Gene-set enrichment analysis of RNA-seq data shows the enrichment of hallmark-IL6-JAK-STAT3-signaling related genes in Mst1/2-deficient NK cells compared to WT NK cells. D Flow cytometry analysis (left) and MFI intensity (right) reveals the expression level of p-STAT3 (Ser727) in NK cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice upon stimulation with or without IL-15 (50 ng/mL) for 30 min ( n = 4). E ChIP-qPCR analysis demonstrated binding of p-STAT3 conserved motifs in the Tcf7 5′ regulatory region (−17.5 kb), while no binding was observed in a region without p-STAT3-binding motifs (+0.2 kb), serving as a negative control. The experiments were using sorted NK cells from WT mice with anti-p-STAT3 antibody or isotype-matched IgG. F – I WT splenocytes were treated with DMSO, Mst1 inhibitor XMU-MP-1 (1 mM, MedChemExpress) or p-STAT3 inhibitor Stattic (1 mM, MedChemExpress) for 24 h, followed by flow cytometry analysis. The representative plots (left) and summary data (right) show the expression level of p-STAT3 (F), TCF1 ( G ), total cellular ROS ( H ) and the percentage of 7AAD + NK cells ( I ) ( n = 4). J Schematic illustration of Mst1/2 regulate NK cell survival and function via controlling metabolic state and transcriptional activity. Data A , B and D – I are representative of three independent experiments with similar results.

    Journal: Cell Death & Disease

    Article Title: Hippo kinases Mst1 and Mst2 maintain NK cell homeostasis by orchestrating metabolic state and transcriptional activity

    doi: 10.1038/s41419-024-06828-x

    Figure Lengend Snippet: A , B Flow cytometry analysis (left) and MFI intensity (right) demonstrate the expression levels of p-STAT5, p-S6, p-AKT473 and p-Erk in NK cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice upon stimulation with or without IL-15 (50 ng/mL) for 30 min ( n ≥ 4). C Gene-set enrichment analysis of RNA-seq data shows the enrichment of hallmark-IL6-JAK-STAT3-signaling related genes in Mst1/2-deficient NK cells compared to WT NK cells. D Flow cytometry analysis (left) and MFI intensity (right) reveals the expression level of p-STAT3 (Ser727) in NK cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice upon stimulation with or without IL-15 (50 ng/mL) for 30 min ( n = 4). E ChIP-qPCR analysis demonstrated binding of p-STAT3 conserved motifs in the Tcf7 5′ regulatory region (−17.5 kb), while no binding was observed in a region without p-STAT3-binding motifs (+0.2 kb), serving as a negative control. The experiments were using sorted NK cells from WT mice with anti-p-STAT3 antibody or isotype-matched IgG. F – I WT splenocytes were treated with DMSO, Mst1 inhibitor XMU-MP-1 (1 mM, MedChemExpress) or p-STAT3 inhibitor Stattic (1 mM, MedChemExpress) for 24 h, followed by flow cytometry analysis. The representative plots (left) and summary data (right) show the expression level of p-STAT3 (F), TCF1 ( G ), total cellular ROS ( H ) and the percentage of 7AAD + NK cells ( I ) ( n = 4). J Schematic illustration of Mst1/2 regulate NK cell survival and function via controlling metabolic state and transcriptional activity. Data A , B and D – I are representative of three independent experiments with similar results.

    Article Snippet: Monoclonal antibodies against mouse TCF1 (Cat#9066S), phospho-Mob1 (Thr35) (Cat#8699), Bim (C34C5) Rabbit mAb (Cat#12186), Phospho-Stat5 (Tyr694) (Cat#9359), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cat#4370S) and polyclonal antibodies against mouse Mst1 (Cat#3682) were obtained from Cell Signaling Technology (Beverly, MA).

    Techniques: Flow Cytometry, Expressing, RNA Sequencing Assay, Binding Assay, Negative Control, Activity Assay

    (A) Schematic of ITA generation by cis -aconitate decarboxylase (Acod1). (B and C) PRs or PRp cells were grown in the indicated conditions for 24 h. (B) Lysates were collected and immunoblotted as noted. (C) Gene expression of Acod1 by quantitative real-time PCR calculated as FC relative to Att PRs. (D) PRp cells were grown in the indicated conditions in the presence of DMSO or carbonyl cyanide m -chlorophenyl hydrazone (CCCP) (20 μM) for 48 h. Lysates were collected and immunoblotted as noted. (E) PRs or PRp cells were grown in the indicated conditions in the presence of DMSO or the nuclear factor κB (NF-κB) inhibitor BAY1170-82 (2.5 μM) for 9 h. Lysates were collected and immunoblotted as noted. Graphs represent data collected from a minimum of 3 biological replicates, and all western blotting experiments were independently repeated a minimum of three times with similar results. p values are calculated by one-way ANOVA followed by Tukey’s test in (C). Data are mean ± SEM.

    Journal: Cell reports

    Article Title: Acod1 expression in cancer cells promotes immune evasion through the generation of inhibitory peptides

    doi: 10.1016/j.celrep.2024.113984

    Figure Lengend Snippet: (A) Schematic of ITA generation by cis -aconitate decarboxylase (Acod1). (B and C) PRs or PRp cells were grown in the indicated conditions for 24 h. (B) Lysates were collected and immunoblotted as noted. (C) Gene expression of Acod1 by quantitative real-time PCR calculated as FC relative to Att PRs. (D) PRp cells were grown in the indicated conditions in the presence of DMSO or carbonyl cyanide m -chlorophenyl hydrazone (CCCP) (20 μM) for 48 h. Lysates were collected and immunoblotted as noted. (E) PRs or PRp cells were grown in the indicated conditions in the presence of DMSO or the nuclear factor κB (NF-κB) inhibitor BAY1170-82 (2.5 μM) for 9 h. Lysates were collected and immunoblotted as noted. Graphs represent data collected from a minimum of 3 biological replicates, and all western blotting experiments were independently repeated a minimum of three times with similar results. p values are calculated by one-way ANOVA followed by Tukey’s test in (C). Data are mean ± SEM.

    Article Snippet: Rabbit polyclonal anti-mouse Irg1/Acod1 , , Cell Signaling , , #17805; RRID: AB_3064865 , , .

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

    (A) Experimental design of in vivo experiment. (B) Western blot for Acod1 of cells injected for tumor experiments. (C) PRp Scr or PRp Acod1 KO cells were subcutaneously injected in mice and received either isotype control (IgG) or αPD-1 monoclonal antibody (mAb) when tumors reached ≥50 mm 3 . Data are mean ± SEM (n = 6–10 per group), and p values were calculated by two-way ANOVA. (D and E) Percentage of CD4 + (D) or CD8 + (E) T cells within each tumor. Data represent the mean ± SEM (n = 4 mice/group), and p values were calculated by two-tailed Student’s t test. n.s., not significant. (F) Representative images of immunofluorescence (IF) staining of GzmB + (red) and TUNEL + (green) cells in tumors from the indicated conditions described in (A). Scale bars of low and high magnification represent 200 and 50 μm, respectively. DAPI, 4′,6-diamidino-2-phenylindole. (G) Quantification of relative GzmB intensity (top) or average number of TUNEL + cells (bottom) with SD from six random fields of view (FOVs). The p values were calculated by one-way ANOVA. n.s., not significant.

    Journal: Cell reports

    Article Title: Acod1 expression in cancer cells promotes immune evasion through the generation of inhibitory peptides

    doi: 10.1016/j.celrep.2024.113984

    Figure Lengend Snippet: (A) Experimental design of in vivo experiment. (B) Western blot for Acod1 of cells injected for tumor experiments. (C) PRp Scr or PRp Acod1 KO cells were subcutaneously injected in mice and received either isotype control (IgG) or αPD-1 monoclonal antibody (mAb) when tumors reached ≥50 mm 3 . Data are mean ± SEM (n = 6–10 per group), and p values were calculated by two-way ANOVA. (D and E) Percentage of CD4 + (D) or CD8 + (E) T cells within each tumor. Data represent the mean ± SEM (n = 4 mice/group), and p values were calculated by two-tailed Student’s t test. n.s., not significant. (F) Representative images of immunofluorescence (IF) staining of GzmB + (red) and TUNEL + (green) cells in tumors from the indicated conditions described in (A). Scale bars of low and high magnification represent 200 and 50 μm, respectively. DAPI, 4′,6-diamidino-2-phenylindole. (G) Quantification of relative GzmB intensity (top) or average number of TUNEL + cells (bottom) with SD from six random fields of view (FOVs). The p values were calculated by one-way ANOVA. n.s., not significant.

    Article Snippet: Rabbit polyclonal anti-mouse Irg1/Acod1 , , Cell Signaling , , #17805; RRID: AB_3064865 , , .

    Techniques: In Vivo, Western Blot, Injection, Two Tailed Test, Immunofluorescence, Staining, TUNEL Assay

    (A) Percentage of EdU + CD8 + T cells following 48 h of activation with αCD3/CD28 in indicated conditioned medium (CM). (B) Representative histograms of violet proliferation dye 450 (VPD450) dilution in CD8 + T cells following 72 h of activation with αCD3/CD28 in indicated CM. (C) Histograms of CD44 (left) and CD25 (right) expression in CD8 + T cells following 72 h of activation with αCD3/CD28 in indicated CM (n = 3). (D) Percentage of CD44 + (left) and CD25 + (right) CD8 + T cells following 72 h of activation with αCD3/CD28 in indicated CM. (E) Cells grown for 24 h in Det conditions. Lysates were collected and immunoblotted as noted. (F) Percentage of EdU + CD8 + T cells as described in (A). (G) Histograms of CD44 (left) and CD25 (right) expression as described in (C). Scr, PRp Scr CM; KO, PRp Acod1 KO CM (n = 3). (H) Percentage of CD44 + (left) and CD25 + (right) CD8 + T cells as described in (D). Data from EdU experiments (A and F) represent the means ± SEM of triplicate wells, and p values were calculated by one-way ANOVA analysis. Data are representative of three independent experiments. The p values for the T cell activation experiments (D and H) were calculated using a paired, two-tailed t test (n = 4 mice/group). n.s., not significant. Western blotting and VPD450 dilution experiments were independently repeated a minimum of three times with similar results.

    Journal: Cell reports

    Article Title: Acod1 expression in cancer cells promotes immune evasion through the generation of inhibitory peptides

    doi: 10.1016/j.celrep.2024.113984

    Figure Lengend Snippet: (A) Percentage of EdU + CD8 + T cells following 48 h of activation with αCD3/CD28 in indicated conditioned medium (CM). (B) Representative histograms of violet proliferation dye 450 (VPD450) dilution in CD8 + T cells following 72 h of activation with αCD3/CD28 in indicated CM. (C) Histograms of CD44 (left) and CD25 (right) expression in CD8 + T cells following 72 h of activation with αCD3/CD28 in indicated CM (n = 3). (D) Percentage of CD44 + (left) and CD25 + (right) CD8 + T cells following 72 h of activation with αCD3/CD28 in indicated CM. (E) Cells grown for 24 h in Det conditions. Lysates were collected and immunoblotted as noted. (F) Percentage of EdU + CD8 + T cells as described in (A). (G) Histograms of CD44 (left) and CD25 (right) expression as described in (C). Scr, PRp Scr CM; KO, PRp Acod1 KO CM (n = 3). (H) Percentage of CD44 + (left) and CD25 + (right) CD8 + T cells as described in (D). Data from EdU experiments (A and F) represent the means ± SEM of triplicate wells, and p values were calculated by one-way ANOVA analysis. Data are representative of three independent experiments. The p values for the T cell activation experiments (D and H) were calculated using a paired, two-tailed t test (n = 4 mice/group). n.s., not significant. Western blotting and VPD450 dilution experiments were independently repeated a minimum of three times with similar results.

    Article Snippet: Rabbit polyclonal anti-mouse Irg1/Acod1 , , Cell Signaling , , #17805; RRID: AB_3064865 , , .

    Techniques: Activation Assay, Expressing, Two Tailed Test, Western Blot

    KEY RESOURCES TABLE

    Journal: Cell reports

    Article Title: Acod1 expression in cancer cells promotes immune evasion through the generation of inhibitory peptides

    doi: 10.1016/j.celrep.2024.113984

    Figure Lengend Snippet: KEY RESOURCES TABLE

    Article Snippet: Rabbit polyclonal anti-mouse Irg1/Acod1 , , Cell Signaling , , #17805; RRID: AB_3064865 , , .

    Techniques: Recombinant, Lysis, Staining, Transfection, In Situ, Cell Isolation, Plasmid Preparation, Software