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pe ps6  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc pe ps6
    Pe Ps6, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 3710 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/pe+ps6+antibodies/Phospho-S6+Ribosomal+Protein+(Ser235%2F236)+Antibody/pm37992572-69-42-44
    Average 96 stars, based on 3710 article reviews
    pe ps6 - by Bioz Stars, 2026-09
    96/100 stars

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    Related Articles

    Comparison:

    Article Title: Exit from germinal center to become quiescent memory B cells depends on metabolic reprograming and provision of a survival signal
    Article Snippet: PE-B220, PE-Cy7-CD138, PE-Cy7-CD38, PacificBlue-CD45.1, PE-CD45.2, biotin-CD69, PerCP-Cy5.5-CD86, BV421-CXCR4, V450-Ki67 antibodies, and BV510-streptavidin were purchased from BioLegend.PE-B220, PE-Cy7-CD138, PE-Cy7-CD38, PacificBlue-CD45.1, PE-CD45.2, biotin-CD69, PerCP-Cy5.5-CD86, BV421-CXCR4, V450-Ki67 antibodies, and BV510-streptavidin were purchased from BioLegend.. PE-pRb and PE-pS6 antibodies were purchased from Cell Signaling. c-Myc antibody was purchased from Abcam.. PE-Bcl2 antibody was purchased from Miltenyi Biotec.PE-Bcl2 antibody was purchased from Miltenyi Biotec.

    Gene Expression:

    Article Title: Exit from germinal center to become quiescent memory B cells depends on metabolic reprograming and provision of a survival signal
    Article Snippet: PE-B220, PE-Cy7-CD138, PE-Cy7-CD38, PacificBlue-CD45.1, PE-CD45.2, biotin-CD69, PerCP-Cy5.5-CD86, BV421-CXCR4, V450-Ki67 antibodies, and BV510-streptavidin were purchased from BioLegend.PE-B220, PE-Cy7-CD138, PE-Cy7-CD38, PacificBlue-CD45.1, PE-CD45.2, biotin-CD69, PerCP-Cy5.5-CD86, BV421-CXCR4, V450-Ki67 antibodies, and BV510-streptavidin were purchased from BioLegend.. PE-pRb and PE-pS6 antibodies were purchased from Cell Signaling. c-Myc antibody was purchased from Abcam.. PE-Bcl2 antibody was purchased from Miltenyi Biotec.PE-Bcl2 antibody was purchased from Miltenyi Biotec.

    RNA Sequencing:

    Article Title: Exit from germinal center to become quiescent memory B cells depends on metabolic reprograming and provision of a survival signal
    Article Snippet: PE-B220, PE-Cy7-CD138, PE-Cy7-CD38, PacificBlue-CD45.1, PE-CD45.2, biotin-CD69, PerCP-Cy5.5-CD86, BV421-CXCR4, V450-Ki67 antibodies, and BV510-streptavidin were purchased from BioLegend.PE-B220, PE-Cy7-CD138, PE-Cy7-CD38, PacificBlue-CD45.1, PE-CD45.2, biotin-CD69, PerCP-Cy5.5-CD86, BV421-CXCR4, V450-Ki67 antibodies, and BV510-streptavidin were purchased from BioLegend.. PE-pRb and PE-pS6 antibodies were purchased from Cell Signaling. c-Myc antibody was purchased from Abcam.. PE-Bcl2 antibody was purchased from Miltenyi Biotec.PE-Bcl2 antibody was purchased from Miltenyi Biotec.

    Flow Cytometry:

    Article Title: Exit from germinal center to become quiescent memory B cells depends on metabolic reprograming and provision of a survival signal
    Article Snippet: PE-B220, PE-Cy7-CD138, PE-Cy7-CD38, PacificBlue-CD45.1, PE-CD45.2, biotin-CD69, PerCP-Cy5.5-CD86, BV421-CXCR4, V450-Ki67 antibodies, and BV510-streptavidin were purchased from BioLegend.PE-B220, PE-Cy7-CD138, PE-Cy7-CD38, PacificBlue-CD45.1, PE-CD45.2, biotin-CD69, PerCP-Cy5.5-CD86, BV421-CXCR4, V450-Ki67 antibodies, and BV510-streptavidin were purchased from BioLegend.. PE-pRb and PE-pS6 antibodies were purchased from Cell Signaling. c-Myc antibody was purchased from Abcam.. PE-Bcl2 antibody was purchased from Miltenyi Biotec.PE-Bcl2 antibody was purchased from Miltenyi Biotec.

    Expressing:

    Article Title: Exit from germinal center to become quiescent memory B cells depends on metabolic reprograming and provision of a survival signal
    Article Snippet: PE-B220, PE-Cy7-CD138, PE-Cy7-CD38, PacificBlue-CD45.1, PE-CD45.2, biotin-CD69, PerCP-Cy5.5-CD86, BV421-CXCR4, V450-Ki67 antibodies, and BV510-streptavidin were purchased from BioLegend.PE-B220, PE-Cy7-CD138, PE-Cy7-CD38, PacificBlue-CD45.1, PE-CD45.2, biotin-CD69, PerCP-Cy5.5-CD86, BV421-CXCR4, V450-Ki67 antibodies, and BV510-streptavidin were purchased from BioLegend.. PE-pRb and PE-pS6 antibodies were purchased from Cell Signaling. c-Myc antibody was purchased from Abcam.. PE-Bcl2 antibody was purchased from Miltenyi Biotec.PE-Bcl2 antibody was purchased from Miltenyi Biotec.

    Fluorescence:

    Article Title: Exit from germinal center to become quiescent memory B cells depends on metabolic reprograming and provision of a survival signal
    Article Snippet: PE-B220, PE-Cy7-CD138, PE-Cy7-CD38, PacificBlue-CD45.1, PE-CD45.2, biotin-CD69, PerCP-Cy5.5-CD86, BV421-CXCR4, V450-Ki67 antibodies, and BV510-streptavidin were purchased from BioLegend.PE-B220, PE-Cy7-CD138, PE-Cy7-CD38, PacificBlue-CD45.1, PE-CD45.2, biotin-CD69, PerCP-Cy5.5-CD86, BV421-CXCR4, V450-Ki67 antibodies, and BV510-streptavidin were purchased from BioLegend.. PE-pRb and PE-pS6 antibodies were purchased from Cell Signaling. c-Myc antibody was purchased from Abcam.. PE-Bcl2 antibody was purchased from Miltenyi Biotec.PE-Bcl2 antibody was purchased from Miltenyi Biotec.

    Control:

    Article Title: Exit from germinal center to become quiescent memory B cells depends on metabolic reprograming and provision of a survival signal
    Article Snippet: PE-B220, PE-Cy7-CD138, PE-Cy7-CD38, PacificBlue-CD45.1, PE-CD45.2, biotin-CD69, PerCP-Cy5.5-CD86, BV421-CXCR4, V450-Ki67 antibodies, and BV510-streptavidin were purchased from BioLegend.PE-B220, PE-Cy7-CD138, PE-Cy7-CD38, PacificBlue-CD45.1, PE-CD45.2, biotin-CD69, PerCP-Cy5.5-CD86, BV421-CXCR4, V450-Ki67 antibodies, and BV510-streptavidin were purchased from BioLegend.. PE-pRb and PE-pS6 antibodies were purchased from Cell Signaling. c-Myc antibody was purchased from Abcam.. PE-Bcl2 antibody was purchased from Miltenyi Biotec.PE-Bcl2 antibody was purchased from Miltenyi Biotec.

    Activity Assay:

    Article Title: Exit from germinal center to become quiescent memory B cells depends on metabolic reprograming and provision of a survival signal
    Article Snippet: PE-B220, PE-Cy7-CD138, PE-Cy7-CD38, PacificBlue-CD45.1, PE-CD45.2, biotin-CD69, PerCP-Cy5.5-CD86, BV421-CXCR4, V450-Ki67 antibodies, and BV510-streptavidin were purchased from BioLegend.PE-B220, PE-Cy7-CD138, PE-Cy7-CD38, PacificBlue-CD45.1, PE-CD45.2, biotin-CD69, PerCP-Cy5.5-CD86, BV421-CXCR4, V450-Ki67 antibodies, and BV510-streptavidin were purchased from BioLegend.. PE-pRb and PE-pS6 antibodies were purchased from Cell Signaling. c-Myc antibody was purchased from Abcam.. PE-Bcl2 antibody was purchased from Miltenyi Biotec.PE-Bcl2 antibody was purchased from Miltenyi Biotec.

    Double Knockout:

    Article Title: Exit from germinal center to become quiescent memory B cells depends on metabolic reprograming and provision of a survival signal
    Article Snippet: PE-B220, PE-Cy7-CD138, PE-Cy7-CD38, PacificBlue-CD45.1, PE-CD45.2, biotin-CD69, PerCP-Cy5.5-CD86, BV421-CXCR4, V450-Ki67 antibodies, and BV510-streptavidin were purchased from BioLegend.PE-B220, PE-Cy7-CD138, PE-Cy7-CD38, PacificBlue-CD45.1, PE-CD45.2, biotin-CD69, PerCP-Cy5.5-CD86, BV421-CXCR4, V450-Ki67 antibodies, and BV510-streptavidin were purchased from BioLegend.. PE-pRb and PE-pS6 antibodies were purchased from Cell Signaling. c-Myc antibody was purchased from Abcam.. PE-Bcl2 antibody was purchased from Miltenyi Biotec.PE-Bcl2 antibody was purchased from Miltenyi Biotec.

    Adoptive Transfer Assay:

    Article Title: Exit from germinal center to become quiescent memory B cells depends on metabolic reprograming and provision of a survival signal
    Article Snippet: PE-B220, PE-Cy7-CD138, PE-Cy7-CD38, PacificBlue-CD45.1, PE-CD45.2, biotin-CD69, PerCP-Cy5.5-CD86, BV421-CXCR4, V450-Ki67 antibodies, and BV510-streptavidin were purchased from BioLegend.PE-B220, PE-Cy7-CD138, PE-Cy7-CD38, PacificBlue-CD45.1, PE-CD45.2, biotin-CD69, PerCP-Cy5.5-CD86, BV421-CXCR4, V450-Ki67 antibodies, and BV510-streptavidin were purchased from BioLegend.. PE-pRb and PE-pS6 antibodies were purchased from Cell Signaling. c-Myc antibody was purchased from Abcam.. PE-Bcl2 antibody was purchased from Miltenyi Biotec.PE-Bcl2 antibody was purchased from Miltenyi Biotec.

    Derivative Assay:

    Article Title: Exit from germinal center to become quiescent memory B cells depends on metabolic reprograming and provision of a survival signal
    Article Snippet: PE-B220, PE-Cy7-CD138, PE-Cy7-CD38, PacificBlue-CD45.1, PE-CD45.2, biotin-CD69, PerCP-Cy5.5-CD86, BV421-CXCR4, V450-Ki67 antibodies, and BV510-streptavidin were purchased from BioLegend.PE-B220, PE-Cy7-CD138, PE-Cy7-CD38, PacificBlue-CD45.1, PE-CD45.2, biotin-CD69, PerCP-Cy5.5-CD86, BV421-CXCR4, V450-Ki67 antibodies, and BV510-streptavidin were purchased from BioLegend.. PE-pRb and PE-pS6 antibodies were purchased from Cell Signaling. c-Myc antibody was purchased from Abcam.. PE-Bcl2 antibody was purchased from Miltenyi Biotec.PE-Bcl2 antibody was purchased from Miltenyi Biotec.

    Labeling:

    Article Title: Exit from germinal center to become quiescent memory B cells depends on metabolic reprograming and provision of a survival signal
    Article Snippet: PE-B220, PE-Cy7-CD138, PE-Cy7-CD38, PacificBlue-CD45.1, PE-CD45.2, biotin-CD69, PerCP-Cy5.5-CD86, BV421-CXCR4, V450-Ki67 antibodies, and BV510-streptavidin were purchased from BioLegend.PE-B220, PE-Cy7-CD138, PE-Cy7-CD38, PacificBlue-CD45.1, PE-CD45.2, biotin-CD69, PerCP-Cy5.5-CD86, BV421-CXCR4, V450-Ki67 antibodies, and BV510-streptavidin were purchased from BioLegend.. PE-pRb and PE-pS6 antibodies were purchased from Cell Signaling. c-Myc antibody was purchased from Abcam.. PE-Bcl2 antibody was purchased from Miltenyi Biotec.PE-Bcl2 antibody was purchased from Miltenyi Biotec.

    Injection:

    Article Title: Exit from germinal center to become quiescent memory B cells depends on metabolic reprograming and provision of a survival signal
    Article Snippet: PE-B220, PE-Cy7-CD138, PE-Cy7-CD38, PacificBlue-CD45.1, PE-CD45.2, biotin-CD69, PerCP-Cy5.5-CD86, BV421-CXCR4, V450-Ki67 antibodies, and BV510-streptavidin were purchased from BioLegend.PE-B220, PE-Cy7-CD138, PE-Cy7-CD38, PacificBlue-CD45.1, PE-CD45.2, biotin-CD69, PerCP-Cy5.5-CD86, BV421-CXCR4, V450-Ki67 antibodies, and BV510-streptavidin were purchased from BioLegend.. PE-pRb and PE-pS6 antibodies were purchased from Cell Signaling. c-Myc antibody was purchased from Abcam.. PE-Bcl2 antibody was purchased from Miltenyi Biotec.PE-Bcl2 antibody was purchased from Miltenyi Biotec.

    Generated:

    Article Title: Exit from germinal center to become quiescent memory B cells depends on metabolic reprograming and provision of a survival signal
    Article Snippet: PE-B220, PE-Cy7-CD138, PE-Cy7-CD38, PacificBlue-CD45.1, PE-CD45.2, biotin-CD69, PerCP-Cy5.5-CD86, BV421-CXCR4, V450-Ki67 antibodies, and BV510-streptavidin were purchased from BioLegend.PE-B220, PE-Cy7-CD138, PE-Cy7-CD38, PacificBlue-CD45.1, PE-CD45.2, biotin-CD69, PerCP-Cy5.5-CD86, BV421-CXCR4, V450-Ki67 antibodies, and BV510-streptavidin were purchased from BioLegend.. PE-pRb and PE-pS6 antibodies were purchased from Cell Signaling. c-Myc antibody was purchased from Abcam.. PE-Bcl2 antibody was purchased from Miltenyi Biotec.PE-Bcl2 antibody was purchased from Miltenyi Biotec.

    Clinical Proteomics:

    Article Title: Exit from germinal center to become quiescent memory B cells depends on metabolic reprograming and provision of a survival signal
    Article Snippet: PE-B220, PE-Cy7-CD138, PE-Cy7-CD38, PacificBlue-CD45.1, PE-CD45.2, biotin-CD69, PerCP-Cy5.5-CD86, BV421-CXCR4, V450-Ki67 antibodies, and BV510-streptavidin were purchased from BioLegend.PE-B220, PE-Cy7-CD138, PE-Cy7-CD38, PacificBlue-CD45.1, PE-CD45.2, biotin-CD69, PerCP-Cy5.5-CD86, BV421-CXCR4, V450-Ki67 antibodies, and BV510-streptavidin were purchased from BioLegend.. PE-pRb and PE-pS6 antibodies were purchased from Cell Signaling. c-Myc antibody was purchased from Abcam.. PE-Bcl2 antibody was purchased from Miltenyi Biotec.PE-Bcl2 antibody was purchased from Miltenyi Biotec.



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    Image Search Results


    (A) Liver weight of mice with doxycycline (DOX)-repressible c-Myc-driven HCC fed with regular or sodium bicarbonate (NaHCO3)-containing drinking water and treated with anti-PD1 antibody (iPD1) or isotype control (IgG). Each dot represents an individual mouse liver weight in the different groups, all determined upon euthanasia at twelve weeks after birth (N = 10, 43, 43, 30, and 31 animals for H2O + DOX, H2O + IgG, NaHCO3 + IgG, H2O + iPD1, and NaHCO3 + iPD1 groups, respectively; unpaired t-test). (B) Representative microscopic images of the mTORC1 target phospho-S6 ribosomal protein (pS6)-stained tumor slices. Scale: 100 μm (inset: 40 μm). (C) Quantification of pS6-positive objects in images in B (N = 5 whole-tumor scans from different animals, one-way ANOVA with multiple comparisons). * p < 0.05, *** p < 0.001.

    Journal: Cancer research communications

    Article Title: Na + /H + -exchanger 1 enhances antitumor activity of engineered NK-92 natural killer cells

    doi: 10.1158/2767-9764.crc-22-0270

    Figure Lengend Snippet: (A) Liver weight of mice with doxycycline (DOX)-repressible c-Myc-driven HCC fed with regular or sodium bicarbonate (NaHCO3)-containing drinking water and treated with anti-PD1 antibody (iPD1) or isotype control (IgG). Each dot represents an individual mouse liver weight in the different groups, all determined upon euthanasia at twelve weeks after birth (N = 10, 43, 43, 30, and 31 animals for H2O + DOX, H2O + IgG, NaHCO3 + IgG, H2O + iPD1, and NaHCO3 + iPD1 groups, respectively; unpaired t-test). (B) Representative microscopic images of the mTORC1 target phospho-S6 ribosomal protein (pS6)-stained tumor slices. Scale: 100 μm (inset: 40 μm). (C) Quantification of pS6-positive objects in images in B (N = 5 whole-tumor scans from different animals, one-way ANOVA with multiple comparisons). * p < 0.05, *** p < 0.001.

    Article Snippet: Cells were then stained with PE-Cy7-conjugated rabbit anti-pS6 (Ser235/Ser236) antibody (Cell Signaling Technology, 34411) or isotype control antibody (Cell Signaling Technology, 97492) at 1:50 dilution for 20 min on ice before analysis.

    Techniques: Control, Staining

    (A) In vitro cytotoxicity of Torin1 (mTOR inhibitor)-treated NK-92 cells against the human melanoma cell line WM3629, relative to the untreated cells (E-T ratio = 3:1, N = 6 wells, one-way ANOVA with multiple comparisons versus untreated). (B) Representative immunoblots of the mTORC1 phosphorylation substrates phospho-p70 S6 kinase (pS6K) and phospho-S6 ribosomal protein (pS6) in constitutively active RHEB (RHEBN153T, hereafter referred to as RHEB)-overexpressing or empty vector (EV) control NK-92 cells treated with pH-controlled media for 6 or 24 hours. Endogenous RHEB is shown as the dim, lower band in the images, where the bright, upper band represents the overexpressed RHEB. (C) In vitro cytotoxicity of RHEB-expressing or EV control NK-92 cells against human melanoma cell lines WM3629 and WM4237 in pH-controlled media (E-T ratio = 3:1, N = 4 wells for each cell line, unpaired t-test). (D) Degranulation of RHEB-overexpressing or EV control NK-92 cells towards the human leukemia cell line K562 in pH-controlled media (E-T ratio = 1:2, N = 3 wells, unpaired t-test). * p < 0.05, ** p < 0.01, *** p < 0.001.

    Journal: Cancer research communications

    Article Title: Na + /H + -exchanger 1 enhances antitumor activity of engineered NK-92 natural killer cells

    doi: 10.1158/2767-9764.crc-22-0270

    Figure Lengend Snippet: (A) In vitro cytotoxicity of Torin1 (mTOR inhibitor)-treated NK-92 cells against the human melanoma cell line WM3629, relative to the untreated cells (E-T ratio = 3:1, N = 6 wells, one-way ANOVA with multiple comparisons versus untreated). (B) Representative immunoblots of the mTORC1 phosphorylation substrates phospho-p70 S6 kinase (pS6K) and phospho-S6 ribosomal protein (pS6) in constitutively active RHEB (RHEBN153T, hereafter referred to as RHEB)-overexpressing or empty vector (EV) control NK-92 cells treated with pH-controlled media for 6 or 24 hours. Endogenous RHEB is shown as the dim, lower band in the images, where the bright, upper band represents the overexpressed RHEB. (C) In vitro cytotoxicity of RHEB-expressing or EV control NK-92 cells against human melanoma cell lines WM3629 and WM4237 in pH-controlled media (E-T ratio = 3:1, N = 4 wells for each cell line, unpaired t-test). (D) Degranulation of RHEB-overexpressing or EV control NK-92 cells towards the human leukemia cell line K562 in pH-controlled media (E-T ratio = 1:2, N = 3 wells, unpaired t-test). * p < 0.05, ** p < 0.01, *** p < 0.001.

    Article Snippet: Cells were then stained with PE-Cy7-conjugated rabbit anti-pS6 (Ser235/Ser236) antibody (Cell Signaling Technology, 34411) or isotype control antibody (Cell Signaling Technology, 97492) at 1:50 dilution for 20 min on ice before analysis.

    Techniques: In Vitro, Western Blot, Phospho-proteomics, Plasmid Preparation, Control, Expressing

    (A) Representative immunoblots assessing mTORC1 substrates pS6K and pS6 in NK-92 cells expressing constitutively active NHE1, NHE1-E262I, or EV treated with pH-controlled media for 6 or 24 hours. At the exposure shown, endogenous NHE1 is barely visible due to low expression. (B) Degranulation of trametinib (MEK inhibitor)-treated NK-92 cells against K562 cells, relative to untreated cells (E-T ratio = 1:2, N = 3 wells, one-way ANOVA with multiple comparisons versus untreated). (C) Representative immunoblots assessing phospho-p44/42 MAPK (pERK1/2) in NK-92 cells expressing NHE1, NHE1-E262I, or EV treated with pH-controlled media for 6 or 24 hours. At the exposure shown, endogenous NHE1 is barely visible due to low expression. (D) Percentage of NHE1-, NHE1-E262I-, or EV-expressing NK-92 cells positive for intracellular pERK1/2 overtime after interacting with K562 cells (E-T ratio = 1:1, N = 3 wells, two-way ANOVA with multiple comparisons). * p < 0.05, *** p < 0.001.

    Journal: Cancer research communications

    Article Title: Na + /H + -exchanger 1 enhances antitumor activity of engineered NK-92 natural killer cells

    doi: 10.1158/2767-9764.crc-22-0270

    Figure Lengend Snippet: (A) Representative immunoblots assessing mTORC1 substrates pS6K and pS6 in NK-92 cells expressing constitutively active NHE1, NHE1-E262I, or EV treated with pH-controlled media for 6 or 24 hours. At the exposure shown, endogenous NHE1 is barely visible due to low expression. (B) Degranulation of trametinib (MEK inhibitor)-treated NK-92 cells against K562 cells, relative to untreated cells (E-T ratio = 1:2, N = 3 wells, one-way ANOVA with multiple comparisons versus untreated). (C) Representative immunoblots assessing phospho-p44/42 MAPK (pERK1/2) in NK-92 cells expressing NHE1, NHE1-E262I, or EV treated with pH-controlled media for 6 or 24 hours. At the exposure shown, endogenous NHE1 is barely visible due to low expression. (D) Percentage of NHE1-, NHE1-E262I-, or EV-expressing NK-92 cells positive for intracellular pERK1/2 overtime after interacting with K562 cells (E-T ratio = 1:1, N = 3 wells, two-way ANOVA with multiple comparisons). * p < 0.05, *** p < 0.001.

    Article Snippet: Cells were then stained with PE-Cy7-conjugated rabbit anti-pS6 (Ser235/Ser236) antibody (Cell Signaling Technology, 34411) or isotype control antibody (Cell Signaling Technology, 97492) at 1:50 dilution for 20 min on ice before analysis.

    Techniques: Western Blot, Expressing

    Systemic buffering negated the effect of anti-PD1 antibody in a mouse hepatocellular carcinoma (HCC) model. A, Liver weight of mice with doxycycline (DOX)-repressible MYC –driven HCC fed with regular or sodium bicarbonate (NaHCO 3 )-containing drinking water and treated with anti-PD1 antibody (iPD1) or isotype control (IgG). Each dot represents an individual mouse liver weight in the different groups, all determined upon euthanasia at 12 weeks after birth ( N = 10, 43, 43, 30, and 31 animals for H 2 O + DOX, H 2 O + IgG, NaHCO 3 + IgG, H 2 O + iPD1, and NaHCO 3 + iPD1 groups, respectively; unpaired t test). B, Representative microscopic images of the mTORC1 target pS6-stained tumor slices. Scale: 100 μm (inset: 40 μm). C, Quantification of pS6-positive objects in images in B ( N = 5 whole-tumor scans from different animals, one-way ANOVA with multiple comparisons). *, P < 0.05; ***, P < 0.001.

    Journal: Cancer Research Communications

    Article Title: Na + /H + -exchanger 1 Enhances Antitumor Activity of Engineered NK-92 Natural Killer Cells

    doi: 10.1158/2767-9764.CRC-22-0270

    Figure Lengend Snippet: Systemic buffering negated the effect of anti-PD1 antibody in a mouse hepatocellular carcinoma (HCC) model. A, Liver weight of mice with doxycycline (DOX)-repressible MYC –driven HCC fed with regular or sodium bicarbonate (NaHCO 3 )-containing drinking water and treated with anti-PD1 antibody (iPD1) or isotype control (IgG). Each dot represents an individual mouse liver weight in the different groups, all determined upon euthanasia at 12 weeks after birth ( N = 10, 43, 43, 30, and 31 animals for H 2 O + DOX, H 2 O + IgG, NaHCO 3 + IgG, H 2 O + iPD1, and NaHCO 3 + iPD1 groups, respectively; unpaired t test). B, Representative microscopic images of the mTORC1 target pS6-stained tumor slices. Scale: 100 μm (inset: 40 μm). C, Quantification of pS6-positive objects in images in B ( N = 5 whole-tumor scans from different animals, one-way ANOVA with multiple comparisons). *, P < 0.05; ***, P < 0.001.

    Article Snippet: Cells were then stained with PE-Cy7–conjugated rabbit anti-pS6 (Ser235/Ser236) antibody (Cell Signaling Technology, 34411) or isotype control antibody (Cell Signaling Technology, 97492) at 1:50 dilution for 20 minutes on ice before analysis.

    Techniques: Control, Staining

    Activating mTORC1 by RHEB enhances cytolytic activity of NK-92 cells. A, In vitro cytotoxicity of Torin1 (mTOR inhibitor)-treated NK-92 cells against the human melanoma cell line WM3629, relative to the untreated cells (E-T ratio = 3:1, N = 6 wells, one-way ANOVA with multiple comparisons versus untreated). B, Representative immunoblots of the mTORC1 phosphorylation substrates pS6K and pS6 in constitutively active RHEB (RHEB N153T , hereafter referred to as RHEB)-overexpressing or EV control NK-92 cells treated with pH-controlled media for 6 or 24 hours. Endogenous RHEB is shown as the dim, lower band in the images, where the bright, upper band represents the overexpressed RHEB. C, In vitro cytotoxicity of RHEB-expressing or EV control NK-92 cells against human melanoma cell lines WM3629 and WM4237 in pH-controlled media (E-T ratio = 3:1, N = 4 wells for each cell line, unpaired t test). D, Degranulation of RHEB-overexpressing or EV control NK-92 cells towards the human leukemia cell line K562 in pH-controlled media (E-T ratio = 1:2, N = 3 wells, unpaired t test). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

    Journal: Cancer Research Communications

    Article Title: Na + /H + -exchanger 1 Enhances Antitumor Activity of Engineered NK-92 Natural Killer Cells

    doi: 10.1158/2767-9764.CRC-22-0270

    Figure Lengend Snippet: Activating mTORC1 by RHEB enhances cytolytic activity of NK-92 cells. A, In vitro cytotoxicity of Torin1 (mTOR inhibitor)-treated NK-92 cells against the human melanoma cell line WM3629, relative to the untreated cells (E-T ratio = 3:1, N = 6 wells, one-way ANOVA with multiple comparisons versus untreated). B, Representative immunoblots of the mTORC1 phosphorylation substrates pS6K and pS6 in constitutively active RHEB (RHEB N153T , hereafter referred to as RHEB)-overexpressing or EV control NK-92 cells treated with pH-controlled media for 6 or 24 hours. Endogenous RHEB is shown as the dim, lower band in the images, where the bright, upper band represents the overexpressed RHEB. C, In vitro cytotoxicity of RHEB-expressing or EV control NK-92 cells against human melanoma cell lines WM3629 and WM4237 in pH-controlled media (E-T ratio = 3:1, N = 4 wells for each cell line, unpaired t test). D, Degranulation of RHEB-overexpressing or EV control NK-92 cells towards the human leukemia cell line K562 in pH-controlled media (E-T ratio = 1:2, N = 3 wells, unpaired t test). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

    Article Snippet: Cells were then stained with PE-Cy7–conjugated rabbit anti-pS6 (Ser235/Ser236) antibody (Cell Signaling Technology, 34411) or isotype control antibody (Cell Signaling Technology, 97492) at 1:50 dilution for 20 minutes on ice before analysis.

    Techniques: Activity Assay, In Vitro, Western Blot, Phospho-proteomics, Control, Expressing

    The increased cytolytic activity of NHE1-expressing NK-92 cells does not correlate with the activation of mTORC1 or ERK pathways. A, Representative immunoblots assessing mTORC1 substrates pS6K and pS6 in NK-92 cells expressing constitutively active NHE1, NHE1-E262I, or EV treated with pH-controlled media for 6 or 24 hours. At the exposure shown, endogenous NHE1 is barely visible due to low expression. B, Degranulation of trametinib (MEK inhibitor)-treated NK-92 cells against K562 cells, relative to untreated cells (E-T ratio = 1:2, N = 3 wells, one-way ANOVA with multiple comparisons vs. untreated). C, Representative immunoblots assessing phospho-p44/42 MAPK (pERK1/2) in NK-92 cells expressing NHE1, NHE1-E262I, or EV treated with pH-controlled media for 6 or 24 hours. At the exposure shown, endogenous NHE1 is barely visible due to low expression. D, Percentage of NHE1-, NHE1-E262I-, or EV-expressing NK-92 cells positive for intracellular pERK1/2 overtime after interacting with K562 cells (E-T ratio = 1:1, N = 3 wells, two-way ANOVA with multiple comparisons). *, P < 0.05; ***, P < 0.001.

    Journal: Cancer Research Communications

    Article Title: Na + /H + -exchanger 1 Enhances Antitumor Activity of Engineered NK-92 Natural Killer Cells

    doi: 10.1158/2767-9764.CRC-22-0270

    Figure Lengend Snippet: The increased cytolytic activity of NHE1-expressing NK-92 cells does not correlate with the activation of mTORC1 or ERK pathways. A, Representative immunoblots assessing mTORC1 substrates pS6K and pS6 in NK-92 cells expressing constitutively active NHE1, NHE1-E262I, or EV treated with pH-controlled media for 6 or 24 hours. At the exposure shown, endogenous NHE1 is barely visible due to low expression. B, Degranulation of trametinib (MEK inhibitor)-treated NK-92 cells against K562 cells, relative to untreated cells (E-T ratio = 1:2, N = 3 wells, one-way ANOVA with multiple comparisons vs. untreated). C, Representative immunoblots assessing phospho-p44/42 MAPK (pERK1/2) in NK-92 cells expressing NHE1, NHE1-E262I, or EV treated with pH-controlled media for 6 or 24 hours. At the exposure shown, endogenous NHE1 is barely visible due to low expression. D, Percentage of NHE1-, NHE1-E262I-, or EV-expressing NK-92 cells positive for intracellular pERK1/2 overtime after interacting with K562 cells (E-T ratio = 1:1, N = 3 wells, two-way ANOVA with multiple comparisons). *, P < 0.05; ***, P < 0.001.

    Article Snippet: Cells were then stained with PE-Cy7–conjugated rabbit anti-pS6 (Ser235/Ser236) antibody (Cell Signaling Technology, 34411) or isotype control antibody (Cell Signaling Technology, 97492) at 1:50 dilution for 20 minutes on ice before analysis.

    Techniques: Activity Assay, Expressing, Activation Assay, Western Blot

    Comparison of gene expression profile between Fr.2 and Fr.5 cells. (A) GSEA of Fr.2 and Fr.5 RNA-seq data . Selected gene sets are shown with normalized enrichment scores (NES) and false discovery rate (FDR) q-values (q-val). (B) Left: Flow cytometry analysis of intracellular (ic) expression of pS6, c-Myc, and pRb in Fr.2, Fr.5, and naive B cells. Right: Cumulative data of geometric mean fluorescence intensity (gMFI). n = 4 (pS6, pRb), n = 3 (c-Myc), representative of two independent experiments. (C) GSEA of Fr.2 and Fr.5 RNA-seq data. All Hallmark gene sets enriched in Fr.2 or Fr.5 (FDR q-value < 0.25) were listed with NES and nominal (NOM) P value (p-val). *, P < 0.05; **, P < 0.01; unpaired Student’s t test.

    Journal: The Journal of Experimental Medicine

    Article Title: Exit from germinal center to become quiescent memory B cells depends on metabolic reprograming and provision of a survival signal

    doi: 10.1084/jem.20200866

    Figure Lengend Snippet: Comparison of gene expression profile between Fr.2 and Fr.5 cells. (A) GSEA of Fr.2 and Fr.5 RNA-seq data . Selected gene sets are shown with normalized enrichment scores (NES) and false discovery rate (FDR) q-values (q-val). (B) Left: Flow cytometry analysis of intracellular (ic) expression of pS6, c-Myc, and pRb in Fr.2, Fr.5, and naive B cells. Right: Cumulative data of geometric mean fluorescence intensity (gMFI). n = 4 (pS6, pRb), n = 3 (c-Myc), representative of two independent experiments. (C) GSEA of Fr.2 and Fr.5 RNA-seq data. All Hallmark gene sets enriched in Fr.2 or Fr.5 (FDR q-value < 0.25) were listed with NES and nominal (NOM) P value (p-val). *, P < 0.05; **, P < 0.01; unpaired Student’s t test.

    Article Snippet: PE-pRb and PE-pS6 antibodies were purchased from Cell Signaling. c-Myc antibody was purchased from Abcam.

    Techniques: Comparison, Gene Expression, RNA Sequencing, Flow Cytometry, Expressing, Fluorescence

    Cellular survival of pro-memory B cells. (A) Flow cytometry analysis of intracellular (ic) expression of pS6 and c-Myc in Fr. 3, Fr.5, and naive B cells (related to ). Representative of three independent experiments. (B) Left: Flow cytometry analysis of donor NP + IgG1 + CD73 + memory B cells. Congenically marked Bcl2l11 +/+ and Bcl2l11 f/+ ERT2cre B1-8 ge naive B cells were cotransferred as a 1:1 mixture into wild-type mice, which were immunized with NP-CGG/alum, administered with tamoxifen on day 8, and analyzed on day 21 (see also ). Right: Cumulative data of Bcl2l11 f/+ : Bcl2l11 +/+ ratio. n = 4, representative of two independent experiments. (C) Top: Flow cytometry analysis of aCasp3 expression in donor IgG1 + LZ B cells (related to ). Bottom: Cumulative data of aCasp3 + ratio. n = 4 (vehicle), n = 7 (tamoxifen), pooled from two independent experiments. (D) Left: Protocol of the control experiment related to . Congenically marked B1-8 ge ERT2cre and Prdm1 f/+ B1-8 ge ERT2cre naive B cells were mixed at a 1:1 ratio and cotransferred into wild-type CD45.1/1 recipient mice. After immunization with NP-CGG/alum, tamoxifen was administered on day 10, and spleens were analyzed on day 12. Right: Flow cytometry analysis of aCasp3 expression in donor NP + IgG1 + CD38 + Efnb1 + (Fr.5/6) B cells. Representative of three independent experiments. **, P < 0.01; unpaired Student’s t test (B) and *, P < 0.05; paired Student’s t test (C). FSC, forward scatter.

    Journal: The Journal of Experimental Medicine

    Article Title: Exit from germinal center to become quiescent memory B cells depends on metabolic reprograming and provision of a survival signal

    doi: 10.1084/jem.20200866

    Figure Lengend Snippet: Cellular survival of pro-memory B cells. (A) Flow cytometry analysis of intracellular (ic) expression of pS6 and c-Myc in Fr. 3, Fr.5, and naive B cells (related to ). Representative of three independent experiments. (B) Left: Flow cytometry analysis of donor NP + IgG1 + CD73 + memory B cells. Congenically marked Bcl2l11 +/+ and Bcl2l11 f/+ ERT2cre B1-8 ge naive B cells were cotransferred as a 1:1 mixture into wild-type mice, which were immunized with NP-CGG/alum, administered with tamoxifen on day 8, and analyzed on day 21 (see also ). Right: Cumulative data of Bcl2l11 f/+ : Bcl2l11 +/+ ratio. n = 4, representative of two independent experiments. (C) Top: Flow cytometry analysis of aCasp3 expression in donor IgG1 + LZ B cells (related to ). Bottom: Cumulative data of aCasp3 + ratio. n = 4 (vehicle), n = 7 (tamoxifen), pooled from two independent experiments. (D) Left: Protocol of the control experiment related to . Congenically marked B1-8 ge ERT2cre and Prdm1 f/+ B1-8 ge ERT2cre naive B cells were mixed at a 1:1 ratio and cotransferred into wild-type CD45.1/1 recipient mice. After immunization with NP-CGG/alum, tamoxifen was administered on day 10, and spleens were analyzed on day 12. Right: Flow cytometry analysis of aCasp3 expression in donor NP + IgG1 + CD38 + Efnb1 + (Fr.5/6) B cells. Representative of three independent experiments. **, P < 0.01; unpaired Student’s t test (B) and *, P < 0.05; paired Student’s t test (C). FSC, forward scatter.

    Article Snippet: PE-pRb and PE-pS6 antibodies were purchased from Cell Signaling. c-Myc antibody was purchased from Abcam.

    Techniques: Flow Cytometry, Expressing, Control

    Hyper mTORC1 activity in Bach2/Blimp1 double-deficient GC B cells. (A) Experimental design of inducible Bach2 and Blimp1 double knockout (dKO) in the adoptive transfer experiment. Bach2 +/+ Prdm1 +/+ , Bach2 +/+ Prdm1 f/f , or Bach2 f/f Prdm1 f/f ERT2cre B1-8 hi naive B cells were independently transferred into wild-type CD45.1 + mice. Mice were administered with tamoxifen, immunized with NP-CGG/alum, and analyzed on day 12. (B) Left: Flow cytometry analysis of donor NP + GC, donor NP + IgG1 + LZ, and donor NP + IgG1 + CD38 + cells. Right: Cumulative data of DZ:LZ ratio, and Fr.2, Fr.5, and Fr.7 cell numbers. Bach2 +/+ Prdm1 +/+ and Bach2 f/f Prdm1 f/f donor-derived NP + IgG1 + GC B cell numbers are 4,206 ± 1,813 and 1,243 ± 374 cells per 10 6 splenocytes, constituted 48 ± 1.3% and 31 ± 3.3% among total recipient GC B cells, respectively. n = 4 ( Bach2 +/+ Prdm1 +/+ ), n = 7 ( Bach2 +/+ Prdm1 f/f ), n = 8 ( Bach2 f/f Prdm1 f/f ) for DZ:LZ ratio, pooled from four independent experiments. n = 5 ( Bach2 +/+ Prdm1 +/+ ), n = 4 ( Bach2 +/+ Prdm1 f/f , Bach2 f/f Prdm1 f/f ) for cell number. Representative of two independent experiments. (C) GSEA of RNA-seq data from LZ B cells. Bach2 +/+ Prdm1 f/f , or Bach2 f/f Prdm1 f/f ERT2cre B1-8 hi naive B cells were independently transferred into wild-type CD45.1 + mice. Mice were administered with tamoxifen and immunized with NP-CGG/alum. NP-specific donor GC LZ and DZ B cells were sorted from five pooled recipients on day 10 for one RNA-seq sample. See also for heatmap of the top 50 differentially expressed genes. n = 2 for each population. Gene sets with false discovery rate (FDR) q-value (q-val) < 0.25 are shown in the table. (D) Left: Flow cytometry analysis of intracellular (ic) expression of c-Myc, pRb, and pS6 in control (Cont; Bach2 +/+ Prdm1 +/+ or Bach2 +/+ Prdm1 f/f ) LZ, dKO ( Bach2 f/f Prdm1 f/f ) LZ, and wild-type naive B cells. Right: Cumulative data of geometric mean fluorescence intensity (gMFI). n = 3, representative of two independent experiments. (E) Top: Flow cytometry analysis of pulse-labeled EdU incorporation in control ( Bach2 +/+ Prdm1 +/+ or Bach2 +/+ Prdm1 f/f ) and dKO ( Bach2 f/f Prdm1 f/f ) GC B cells. Bottom: Cumulative data of EdU + ratio. n = 3, representative of two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; unpaired Student’s t test.

    Journal: The Journal of Experimental Medicine

    Article Title: Exit from germinal center to become quiescent memory B cells depends on metabolic reprograming and provision of a survival signal

    doi: 10.1084/jem.20200866

    Figure Lengend Snippet: Hyper mTORC1 activity in Bach2/Blimp1 double-deficient GC B cells. (A) Experimental design of inducible Bach2 and Blimp1 double knockout (dKO) in the adoptive transfer experiment. Bach2 +/+ Prdm1 +/+ , Bach2 +/+ Prdm1 f/f , or Bach2 f/f Prdm1 f/f ERT2cre B1-8 hi naive B cells were independently transferred into wild-type CD45.1 + mice. Mice were administered with tamoxifen, immunized with NP-CGG/alum, and analyzed on day 12. (B) Left: Flow cytometry analysis of donor NP + GC, donor NP + IgG1 + LZ, and donor NP + IgG1 + CD38 + cells. Right: Cumulative data of DZ:LZ ratio, and Fr.2, Fr.5, and Fr.7 cell numbers. Bach2 +/+ Prdm1 +/+ and Bach2 f/f Prdm1 f/f donor-derived NP + IgG1 + GC B cell numbers are 4,206 ± 1,813 and 1,243 ± 374 cells per 10 6 splenocytes, constituted 48 ± 1.3% and 31 ± 3.3% among total recipient GC B cells, respectively. n = 4 ( Bach2 +/+ Prdm1 +/+ ), n = 7 ( Bach2 +/+ Prdm1 f/f ), n = 8 ( Bach2 f/f Prdm1 f/f ) for DZ:LZ ratio, pooled from four independent experiments. n = 5 ( Bach2 +/+ Prdm1 +/+ ), n = 4 ( Bach2 +/+ Prdm1 f/f , Bach2 f/f Prdm1 f/f ) for cell number. Representative of two independent experiments. (C) GSEA of RNA-seq data from LZ B cells. Bach2 +/+ Prdm1 f/f , or Bach2 f/f Prdm1 f/f ERT2cre B1-8 hi naive B cells were independently transferred into wild-type CD45.1 + mice. Mice were administered with tamoxifen and immunized with NP-CGG/alum. NP-specific donor GC LZ and DZ B cells were sorted from five pooled recipients on day 10 for one RNA-seq sample. See also for heatmap of the top 50 differentially expressed genes. n = 2 for each population. Gene sets with false discovery rate (FDR) q-value (q-val) < 0.25 are shown in the table. (D) Left: Flow cytometry analysis of intracellular (ic) expression of c-Myc, pRb, and pS6 in control (Cont; Bach2 +/+ Prdm1 +/+ or Bach2 +/+ Prdm1 f/f ) LZ, dKO ( Bach2 f/f Prdm1 f/f ) LZ, and wild-type naive B cells. Right: Cumulative data of geometric mean fluorescence intensity (gMFI). n = 3, representative of two independent experiments. (E) Top: Flow cytometry analysis of pulse-labeled EdU incorporation in control ( Bach2 +/+ Prdm1 +/+ or Bach2 +/+ Prdm1 f/f ) and dKO ( Bach2 f/f Prdm1 f/f ) GC B cells. Bottom: Cumulative data of EdU + ratio. n = 3, representative of two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; unpaired Student’s t test.

    Article Snippet: PE-pRb and PE-pS6 antibodies were purchased from Cell Signaling. c-Myc antibody was purchased from Abcam.

    Techniques: Activity Assay, Double Knockout, Adoptive Transfer Assay, Flow Cytometry, Derivative Assay, RNA Sequencing, Expressing, Control, Fluorescence, Labeling

    Partial rescue of memory B cell generation by rapamycin treatment in Bach2/Blimp1 double-deficient GC B cells. (A) Experimental design of B cell–specific rapamycin treatment using Bach2 and Blimp1 double-deficient B cells. Control ( Bach2 +/+ Prdm1 +/+ ) or double knockout (dKO; Bach2 f/f Prdm1 f/f ) ERT2cre B1-8 hi CD45.1 + naive B cells were independently transferred into wild-type mice. Mice were administered with tamoxifen, immunized with NP-CGG/alum, injected with rapamycin daily during days 4–11, and analyzed on day 12. (B) Flow cytometry analysis of intracellular (ic) expression of pS6 and c-Myc in control LZ, dKO LZ, and wild-type naive B cells (left), and pulse-labeled EdU incorporation in control and dKO GC B cells (right). Representative of three independent experiments. (C) Left: Flow cytometry analysis of donor NP + and donor NP + GC B cells. Right: Cumulative data of GC, IgG1 + memory, and IgG1 + CD73 + memory B cell number, and DZ:LZ ratio. n = 6–8 for GC and IgG1 + memory B cell number and DZ:LZ ratio, n = 3–5 for IgG1 + CD73 + memory B cell number. Pooled from two independent experiments. (D) Left: Flow cytometry analysis of donor NP + IgG1 + LZ and donor NP + IgG1 + CD38 + B cells. Right: Cumulative data for the Fr.5:Fr.2 ratio. n = 4–6, representative of two independent experiments. *, P < 0.05; **, P < 0.01; unpaired Student’s t test.

    Journal: The Journal of Experimental Medicine

    Article Title: Exit from germinal center to become quiescent memory B cells depends on metabolic reprograming and provision of a survival signal

    doi: 10.1084/jem.20200866

    Figure Lengend Snippet: Partial rescue of memory B cell generation by rapamycin treatment in Bach2/Blimp1 double-deficient GC B cells. (A) Experimental design of B cell–specific rapamycin treatment using Bach2 and Blimp1 double-deficient B cells. Control ( Bach2 +/+ Prdm1 +/+ ) or double knockout (dKO; Bach2 f/f Prdm1 f/f ) ERT2cre B1-8 hi CD45.1 + naive B cells were independently transferred into wild-type mice. Mice were administered with tamoxifen, immunized with NP-CGG/alum, injected with rapamycin daily during days 4–11, and analyzed on day 12. (B) Flow cytometry analysis of intracellular (ic) expression of pS6 and c-Myc in control LZ, dKO LZ, and wild-type naive B cells (left), and pulse-labeled EdU incorporation in control and dKO GC B cells (right). Representative of three independent experiments. (C) Left: Flow cytometry analysis of donor NP + and donor NP + GC B cells. Right: Cumulative data of GC, IgG1 + memory, and IgG1 + CD73 + memory B cell number, and DZ:LZ ratio. n = 6–8 for GC and IgG1 + memory B cell number and DZ:LZ ratio, n = 3–5 for IgG1 + CD73 + memory B cell number. Pooled from two independent experiments. (D) Left: Flow cytometry analysis of donor NP + IgG1 + LZ and donor NP + IgG1 + CD38 + B cells. Right: Cumulative data for the Fr.5:Fr.2 ratio. n = 4–6, representative of two independent experiments. *, P < 0.05; **, P < 0.01; unpaired Student’s t test.

    Article Snippet: PE-pRb and PE-pS6 antibodies were purchased from Cell Signaling. c-Myc antibody was purchased from Abcam.

    Techniques: Control, Double Knockout, Injection, Flow Cytometry, Expressing, Labeling

    Lower mTORC1 activity in GC B cells favors the memory fate. (A) Left: Experimental design of competitive cotransfer of rapamycin-sensitive and -resistant B1-8 ge B cells. Congenically marked Mtor +/+ and Mtor F2108L/F2108L B1-8 ge naive B cells were cotransferred as a 1:1 mixture into Mtor F2108L/F2108L hosts, which were immunized with NP-CGG/alum, administered with rapamycin daily during day 4–13, and analyzed on day 14. Right: Flow cytometry analysis of intracellular (ic) expression of pS6 in Mtor +/+ or Mtor F2108L/F2108L LZ B cells with or without rapamycin treatment. Representative of three independent experiments. (B) Flow cytometry analysis and cumulative data of donor NP + GC (left) and donor NP + memory B cells (right). n = 4, representative of two independent experiments. (C) Experimental design of competitive cotransfer of rapamycin-sensitive and -resistant B1-8 ge B cells with EdU labeling. Mice were prepared as in A, and proliferative GC-derived cells were labeled with EdU (injected i.p. on day 10 and then in the drinking water during days 10–13). (D) Left: Flow cytometry of donor NP + cells prepared as in C. Right: Cumulative data of EdU + Fr.7 memory B cell number and EdU + Fr.7:GC ratio. n = 4, representative of two independent experiments. (E) Left: Flow cytometry of Fr.2 and Fr.5 cells prepared as in C. Right: Cumulative data of Fr.5:Fr.2 ratio. n = 4, representative of two independent experiments. (F) Recall potential of IgG1 + memory B cells generated after rapamycin treatment. B1-8 ge B cells were transferred into Mtor F2108L recipient mice. After immunization with NP-CGG/alum, mice were treated with or without rapamycin as in C, and NP + IgG1 + memory B cells were sorted on day 14. Adoptive transfer with activated CD4 + T cells was performed as in . On day 6 after a boost with NP-CGG, donor NP + splenocytes were analyzed by flow cytometry (top), and donor NP + B cells and donor NP + plasma cells were quantified (bottom). n = 3 (vehicle), n = 4 (rapamycin), pooled from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; paired Student’s t test.

    Journal: The Journal of Experimental Medicine

    Article Title: Exit from germinal center to become quiescent memory B cells depends on metabolic reprograming and provision of a survival signal

    doi: 10.1084/jem.20200866

    Figure Lengend Snippet: Lower mTORC1 activity in GC B cells favors the memory fate. (A) Left: Experimental design of competitive cotransfer of rapamycin-sensitive and -resistant B1-8 ge B cells. Congenically marked Mtor +/+ and Mtor F2108L/F2108L B1-8 ge naive B cells were cotransferred as a 1:1 mixture into Mtor F2108L/F2108L hosts, which were immunized with NP-CGG/alum, administered with rapamycin daily during day 4–13, and analyzed on day 14. Right: Flow cytometry analysis of intracellular (ic) expression of pS6 in Mtor +/+ or Mtor F2108L/F2108L LZ B cells with or without rapamycin treatment. Representative of three independent experiments. (B) Flow cytometry analysis and cumulative data of donor NP + GC (left) and donor NP + memory B cells (right). n = 4, representative of two independent experiments. (C) Experimental design of competitive cotransfer of rapamycin-sensitive and -resistant B1-8 ge B cells with EdU labeling. Mice were prepared as in A, and proliferative GC-derived cells were labeled with EdU (injected i.p. on day 10 and then in the drinking water during days 10–13). (D) Left: Flow cytometry of donor NP + cells prepared as in C. Right: Cumulative data of EdU + Fr.7 memory B cell number and EdU + Fr.7:GC ratio. n = 4, representative of two independent experiments. (E) Left: Flow cytometry of Fr.2 and Fr.5 cells prepared as in C. Right: Cumulative data of Fr.5:Fr.2 ratio. n = 4, representative of two independent experiments. (F) Recall potential of IgG1 + memory B cells generated after rapamycin treatment. B1-8 ge B cells were transferred into Mtor F2108L recipient mice. After immunization with NP-CGG/alum, mice were treated with or without rapamycin as in C, and NP + IgG1 + memory B cells were sorted on day 14. Adoptive transfer with activated CD4 + T cells was performed as in . On day 6 after a boost with NP-CGG, donor NP + splenocytes were analyzed by flow cytometry (top), and donor NP + B cells and donor NP + plasma cells were quantified (bottom). n = 3 (vehicle), n = 4 (rapamycin), pooled from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; paired Student’s t test.

    Article Snippet: PE-pRb and PE-pS6 antibodies were purchased from Cell Signaling. c-Myc antibody was purchased from Abcam.

    Techniques: Activity Assay, Flow Cytometry, Expressing, Labeling, Derivative Assay, Injection, Generated, Adoptive Transfer Assay, Clinical Proteomics

    Obesity increases Th17 cell glycolysis and adiponectin dampens Th17- glycolysis in an AMPK dependent manner. (A) Plasma adiponectin levels in normal control diet (NCD) and high fat diet (HFD) fed mice. Western blot analysis of phospho-AMPK was determined. CD4+ T cells from NCD and HFD fed mice were stimulated with anti-CD3/CD28 for 15 min and expression of phospho-AMPK was measured (B) . (C) MFI of pS6 on CD4+ T cells in NCD and HFD mice following 30 min culture with anti-CD3/CD28. (D–F) Relative messenger RNA expression of glycolytic enzymes hk1 (D) , ldh-a (E) , and pkm (F) in purified Th17 cells in NCD and HFD mice. (G,H) Extracellular acidification rate was measured by seahorse in differentiated Th17 cells from HFD mice and the response to adiponectin and compound C were recorded. (I) Naïve CD4+ T cells were differentiated into Th17 cells in the presence of adiponectin and frequencies of IL-17+ Th17 cells were measured. Two-tailed non-parametric Mann–Whitney U -test was performed for statistical analysis.

    Journal: Frontiers in Immunology

    Article Title: Adiponectin Limits IFN-γ and IL-17 Producing CD4 T Cells in Obesity by Restraining Cell Intrinsic Glycolysis

    doi: 10.3389/fimmu.2019.02555

    Figure Lengend Snippet: Obesity increases Th17 cell glycolysis and adiponectin dampens Th17- glycolysis in an AMPK dependent manner. (A) Plasma adiponectin levels in normal control diet (NCD) and high fat diet (HFD) fed mice. Western blot analysis of phospho-AMPK was determined. CD4+ T cells from NCD and HFD fed mice were stimulated with anti-CD3/CD28 for 15 min and expression of phospho-AMPK was measured (B) . (C) MFI of pS6 on CD4+ T cells in NCD and HFD mice following 30 min culture with anti-CD3/CD28. (D–F) Relative messenger RNA expression of glycolytic enzymes hk1 (D) , ldh-a (E) , and pkm (F) in purified Th17 cells in NCD and HFD mice. (G,H) Extracellular acidification rate was measured by seahorse in differentiated Th17 cells from HFD mice and the response to adiponectin and compound C were recorded. (I) Naïve CD4+ T cells were differentiated into Th17 cells in the presence of adiponectin and frequencies of IL-17+ Th17 cells were measured. Two-tailed non-parametric Mann–Whitney U -test was performed for statistical analysis.

    Article Snippet: CD4-PE-Cy7 and pS6-PE antibodies were purchased from eBioscience and all the other antibodies were procured from Biolegend (Fell, Germany).

    Techniques: Western Blot, Expressing, RNA Expression, Purification, Two Tailed Test, MANN-WHITNEY