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Thermo Fisher ps6 pe antibodies

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 <t>pS6</t> 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.

Ps6 Pe Antibodies, supplied by Thermo Fisher, 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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Images

1) Product Images from "Adiponectin Limits IFN-γ and IL-17 Producing CD4 T Cells in Obesity by Restraining Cell Intrinsic Glycolysis"

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

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2019.02555

Figure Legend 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.

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

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.

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.

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.

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.

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

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

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

( a–d ) Cultured NK cells (6 days in IL15 10 ng/mL) were purified and then co-cultured with or without B16 melanoma cells at an E:T ratio of 2:1 ( a ), with B16 melanoma cells at an E:T ratio of 4:1, 2:1 or 1:2 ( b ), with or without YAC-1, CT26 and LLC tumour cells at an E:T ratio of 1:4 ( c ), or with or without RMA/RMA-S cells at an E:T ratio of 1:4 ( d ) for 18 h before analysis of CD25 expression by flow cytometry. ( e – k ) Cultured NK cells (6 days in IL15 10 ng/mL) were purified and then co-cultured with B16 melanoma cells for 18 h at an E:T ratio of 2:1, washed and put back into culture with IL15 (7.5 ng/mL) with or without ± IL2 (20 ng/mL) for 18 h before analysis by flow cytometry. ( e ) IFNγ production by CD25 high and CD25 neg NK cells cultured in IL2 alone, with B16 cells or with B16 cells plus IL2. ( f ) IFNγ production in CD25 high NK cells cultured with B16 ± IL2. ( g , h ) Analysis of NK cells cultured with B16 cells + IL2 comparing CD25 high and CD25 neg NK cells (g, left panel) for effector functions, IFNγ production and granzyme (Gnzb) B expression. ( i , j ) Analysis of cell size (FSC), cMyc and CD71 expression, and levels of phosphorylated S6 ribosomal protein (pS6) in CD25 high and CD25 neg NK cells from B16 cells + IL2 co-cultures. ( k,l ) Rates of fluorescent transferrin uptake were measured in CD25 high and CD25 neg NK cells from B16 cells + IL2 co-cultures. Data is representative ( a , c , d , e , g , i , k ) or mean ± SEM (b,f,h,j,l) of 3–5 independent experiments. Data was analyzed using a paired students t -test. (* p < 0.05, ** p < 0.01, *** p < 0.001).

Journal: Immunometabolism

Article Title: Natural Killer Cells Integrate Signals Received from Tumour Interactions and IL2 to Induce Robust and Prolonged Anti-Tumour and Metabolic Responses

doi: 10.20900/immunometab20190014

Figure Lengend Snippet: ( a–d ) Cultured NK cells (6 days in IL15 10 ng/mL) were purified and then co-cultured with or without B16 melanoma cells at an E:T ratio of 2:1 ( a ), with B16 melanoma cells at an E:T ratio of 4:1, 2:1 or 1:2 ( b ), with or without YAC-1, CT26 and LLC tumour cells at an E:T ratio of 1:4 ( c ), or with or without RMA/RMA-S cells at an E:T ratio of 1:4 ( d ) for 18 h before analysis of CD25 expression by flow cytometry. ( e – k ) Cultured NK cells (6 days in IL15 10 ng/mL) were purified and then co-cultured with B16 melanoma cells for 18 h at an E:T ratio of 2:1, washed and put back into culture with IL15 (7.5 ng/mL) with or without ± IL2 (20 ng/mL) for 18 h before analysis by flow cytometry. ( e ) IFNγ production by CD25 high and CD25 neg NK cells cultured in IL2 alone, with B16 cells or with B16 cells plus IL2. ( f ) IFNγ production in CD25 high NK cells cultured with B16 ± IL2. ( g , h ) Analysis of NK cells cultured with B16 cells + IL2 comparing CD25 high and CD25 neg NK cells (g, left panel) for effector functions, IFNγ production and granzyme (Gnzb) B expression. ( i , j ) Analysis of cell size (FSC), cMyc and CD71 expression, and levels of phosphorylated S6 ribosomal protein (pS6) in CD25 high and CD25 neg NK cells from B16 cells + IL2 co-cultures. ( k,l ) Rates of fluorescent transferrin uptake were measured in CD25 high and CD25 neg NK cells from B16 cells + IL2 co-cultures. Data is representative ( a , c , d , e , g , i , k ) or mean ± SEM (b,f,h,j,l) of 3–5 independent experiments. Data was analyzed using a paired students t -test. (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: After 30 min incubation, the cells were washed, and stained for extracellular antibodies, along with a secondary antibody for pS6 (PE, donkey anti-rabbit, Jackson Immunoresearch, Cambridgeshire, UK).

Techniques: Cell Culture, Purification, Expressing, Flow Cytometry

( a–e ) Cultured NK cells (6 days in IL15 10 ng/mL) were purified and stimulated with a plate bound α-NK1.1 antibody (10 μg/mL) in media supplemented with low dose IL15 (5 ng/mL) ± IL2 (20 ng/mL) ± rapamycin (20 nM) as indicated, or left unstimulated (IL15; 5 ng/mL). Cells were analysed by immunoblot analysis ( a ), by flow cytometry for levels of phosphorylated S6 ribosomal protein (pS6) ( b ), or the expression of CD71 and CD98 ( e ). Alternatively, rates of OXPHOS ( c ) or glycolysis (d) were measured using the seahorse extracellular flux analyser. Data is representative ( a–e ) or mean ± SEM ( b–d ) of 5–6 independent experiments. Data was analyzed using a one way ANOVA and tukey post test ( b ), a paired students t -test ( c,d ) (* p < 0.05, ** p < 0.01).

Journal: Immunometabolism

Article Title: Natural Killer Cells Integrate Signals Received from Tumour Interactions and IL2 to Induce Robust and Prolonged Anti-Tumour and Metabolic Responses

doi: 10.20900/immunometab20190014

Figure Lengend Snippet: ( a–e ) Cultured NK cells (6 days in IL15 10 ng/mL) were purified and stimulated with a plate bound α-NK1.1 antibody (10 μg/mL) in media supplemented with low dose IL15 (5 ng/mL) ± IL2 (20 ng/mL) ± rapamycin (20 nM) as indicated, or left unstimulated (IL15; 5 ng/mL). Cells were analysed by immunoblot analysis ( a ), by flow cytometry for levels of phosphorylated S6 ribosomal protein (pS6) ( b ), or the expression of CD71 and CD98 ( e ). Alternatively, rates of OXPHOS ( c ) or glycolysis (d) were measured using the seahorse extracellular flux analyser. Data is representative ( a–e ) or mean ± SEM ( b–d ) of 5–6 independent experiments. Data was analyzed using a one way ANOVA and tukey post test ( b ), a paired students t -test ( c,d ) (* p < 0.05, ** p < 0.01).

Techniques: Cell Culture, Purification, Western Blot, Flow Cytometry, Expressing

(a) Cultured NK cells (3 days in IL15 10 ng/mL) were purified and stimulated with a plate bound α-NK1.1 antibody (10 μg/mL) ± IL2 (20 ng/mL) for 24–72 h and analysed by flow cytometry for cell viability. ( b–c ) Cultured NK cells (6 days in IL15 10 ng/mL) were purified and then co-cultured with B16 melanoma cells or for 18 h, washed and put back into culture with IL2 (20 ng/mL) ± rapamycin for 24 h before analysis of CD25 high NK cells by flow cytometry for phosphorylated S6 ribosomal protein (pS6), IFNγ production and granzyme B expression. ( d–g ) Cultured (6 days in IL15 10 ng/mL) from cMyc fl/fl Tamox-Cre and cMyc wt/wt Tamox-Cre were treated with tamoxifen (0.6 μM) and then co-cultured with B16 melanoma cells with IL2 (20 ng/mL) for 18 h before analysis by flow cytometry for cell size and the expression of CD25, cell size, CD71, granzyme B and granzyme B mRNA by rtPCR ( g ). ( h,i ) Cultured NK cells (6 days in IL15 10 ng/mL) were purified and then co-cultured with B16 melanoma cells or for 18 h, washed and put back into culture with IL2 (20 ng/mL) ± BCH (25 mM), ± rapamycin (20 nM) as indicated for 24 h before analysis of CD25 high NK cells by flow cytometry for phosphorylated S6 ribosomal protein (pS6), cMyc and cell viability. Data is representative ( b,e,f,g,h,i ) or mean ± SEM ( a,c,d,f,g,i ) of 3–5 independent experiments. Data was analyzed using a one way ANOVA with a tukey post test or a paired students t -test. (** p < 0.01, *** p < 0.001).

Journal: Immunometabolism

Article Title: Natural Killer Cells Integrate Signals Received from Tumour Interactions and IL2 to Induce Robust and Prolonged Anti-Tumour and Metabolic Responses

doi: 10.20900/immunometab20190014

Figure Lengend Snippet: (a) Cultured NK cells (3 days in IL15 10 ng/mL) were purified and stimulated with a plate bound α-NK1.1 antibody (10 μg/mL) ± IL2 (20 ng/mL) for 24–72 h and analysed by flow cytometry for cell viability. ( b–c ) Cultured NK cells (6 days in IL15 10 ng/mL) were purified and then co-cultured with B16 melanoma cells or for 18 h, washed and put back into culture with IL2 (20 ng/mL) ± rapamycin for 24 h before analysis of CD25 high NK cells by flow cytometry for phosphorylated S6 ribosomal protein (pS6), IFNγ production and granzyme B expression. ( d–g ) Cultured (6 days in IL15 10 ng/mL) from cMyc fl/fl Tamox-Cre and cMyc wt/wt Tamox-Cre were treated with tamoxifen (0.6 μM) and then co-cultured with B16 melanoma cells with IL2 (20 ng/mL) for 18 h before analysis by flow cytometry for cell size and the expression of CD25, cell size, CD71, granzyme B and granzyme B mRNA by rtPCR ( g ). ( h,i ) Cultured NK cells (6 days in IL15 10 ng/mL) were purified and then co-cultured with B16 melanoma cells or for 18 h, washed and put back into culture with IL2 (20 ng/mL) ± BCH (25 mM), ± rapamycin (20 nM) as indicated for 24 h before analysis of CD25 high NK cells by flow cytometry for phosphorylated S6 ribosomal protein (pS6), cMyc and cell viability. Data is representative ( b,e,f,g,h,i ) or mean ± SEM ( a,c,d,f,g,i ) of 3–5 independent experiments. Data was analyzed using a one way ANOVA with a tukey post test or a paired students t -test. (** p < 0.01, *** p < 0.001).

Techniques: Cell Culture, Purification, Flow Cytometry, Expressing, Reverse Transcription Polymerase Chain Reaction

(A) Quantitative real-time (qRT)-PCR analysis of PI3KCD transcripts in peripheral blood mononuclear cell (PBMC) isolated from patients with primary Sjögren’s syndrome (pSS). CD3+ cells (dark grey bar), CD19+ cells (black bar), CD138+ cells (red bar), CD11c+CD11b+ cells (light grey bar). Results represented as mean±SD of five patients; **p<0.01, one-way analysis of variance (ANOVA). (B) qRT-PCR analysis of PI3KCD transcripts in total mRNA isolated from salivary glands of patients with pSS (black circles) and sicca controls (open circles). Results represented as mean±SD of 15–17 patients in each group; *p<0.05, unpaired t-test. (C) Correlation between focus scores (FSC) and levels of PI3KDC expressed as 2^-DCT detected in frozen salivary galnds from patients with pSS. R 2 0.3941, p=0.0092. (D) qRT-PCR analysis of PI3KCD transcripts in microdissected epithelium, foci, germinal centre positive (GC+) foci from salivary glands of patients with pSS and GCs isolated from tonsils. Results represented as mean±SD of 5–10 biological replicates in each category; **p<0.01, ****p<0.0001, one-way ANOVA. (E) Microphotograph of minor salivary glands from patients with pSS, showing in red CD45 staining and in green PI3KCD RNA (visualised with RNAScope). (F) Representative microphotograph of salivary glands from non-specific sialoadenitis control (NSCS) patients stained for the PI3Kδ pathway activation marker phosphorylated ribosomal protein S6 (pS6; green) and 4′,6-diamidino-2-phenylindole (DAPI; grey); scale bars=100 µm. (G) Representative microphotograph of salivary glands from patients with pSS with pS6 (green) and DAPI (grey). (H) Representative microphotographs showing pS6 (green) expression within CD20 (blue) and CD3 or CD138 (red) cells in salivary glands from patients with pSS; scale bars=100 µm. (I) Representative histogram showing flow cytometry staining for pS6 (green) and isotype control (grey) in CD45+ cells present in salivary glands of patients with pSS. viSNE plots of flow cytometry of pSS salivary gland CD45+pS6+ cells. Colours indicate cell expression level of labelled marker. Histogram showing pAkt expression in CD45+pS6+ cells.

Journal: Annals of the Rheumatic Diseases

Article Title: Phosphatidylinositol 3-kinase delta pathway: a novel therapeutic target for Sjögren’s syndrome

doi: 10.1136/annrheumdis-2017-212619

Figure Lengend Snippet: (A) Quantitative real-time (qRT)-PCR analysis of PI3KCD transcripts in peripheral blood mononuclear cell (PBMC) isolated from patients with primary Sjögren’s syndrome (pSS). CD3+ cells (dark grey bar), CD19+ cells (black bar), CD138+ cells (red bar), CD11c+CD11b+ cells (light grey bar). Results represented as mean±SD of five patients; **p<0.01, one-way analysis of variance (ANOVA). (B) qRT-PCR analysis of PI3KCD transcripts in total mRNA isolated from salivary glands of patients with pSS (black circles) and sicca controls (open circles). Results represented as mean±SD of 15–17 patients in each group; *p<0.05, unpaired t-test. (C) Correlation between focus scores (FSC) and levels of PI3KDC expressed as 2^-DCT detected in frozen salivary galnds from patients with pSS. R 2 0.3941, p=0.0092. (D) qRT-PCR analysis of PI3KCD transcripts in microdissected epithelium, foci, germinal centre positive (GC+) foci from salivary glands of patients with pSS and GCs isolated from tonsils. Results represented as mean±SD of 5–10 biological replicates in each category; **p<0.01, ****p<0.0001, one-way ANOVA. (E) Microphotograph of minor salivary glands from patients with pSS, showing in red CD45 staining and in green PI3KCD RNA (visualised with RNAScope). (F) Representative microphotograph of salivary glands from non-specific sialoadenitis control (NSCS) patients stained for the PI3Kδ pathway activation marker phosphorylated ribosomal protein S6 (pS6; green) and 4′,6-diamidino-2-phenylindole (DAPI; grey); scale bars=100 µm. (G) Representative microphotograph of salivary glands from patients with pSS with pS6 (green) and DAPI (grey). (H) Representative microphotographs showing pS6 (green) expression within CD20 (blue) and CD3 or CD138 (red) cells in salivary glands from patients with pSS; scale bars=100 µm. (I) Representative histogram showing flow cytometry staining for pS6 (green) and isotype control (grey) in CD45+ cells present in salivary glands of patients with pSS. viSNE plots of flow cytometry of pSS salivary gland CD45+pS6+ cells. Colours indicate cell expression level of labelled marker. Histogram showing pAkt expression in CD45+pS6+ cells.

Article Snippet: Intracellular staining for Ki67 clone B56 (BD Biosciences) and pS6 PE and pAKT Alexa Fluor 488 (Cell signalling) was performed by using the Cytofix/Perm kit (BD biosciences) and Fixation/Permeabilization Buffer set (ebiosciences) according to the manufacturer’s protocol.

Techniques: Quantitative RT-PCR, Isolation, Staining, RNAscope, Control, Activation Assay, Marker, Expressing, Flow Cytometry

(A) Quantitative real-time PCR analysis of PI3KCD transcripts isolated cells from salivary glands of cannulated mice at day 15 postcannulation (pc). B cells (black bar), T cells (dark grey bar), plasma cells (red bar), macrophages (blue) and dendritic cells (light grey bar), CD45− cells (light yellow bars). Results represented as mean±SD from five mice; *p<0.5, ***p<0.001, one-way analysis of variance. (B) Pie chart showing distribution of different leucocyte populations within CD45+ phosphorylated ribosomal protein S6 (pS6+) cells present in salivary glands of wild-type (WT) mice at day 15 pc (C) viSNE plots of flow cytometry of day 15 pc salivary gland CD45+pS6+ cells. Colour indicates cell expression level of labelled marker. Data is representative of two independent experiments with five mice. (D) Histogram showing phosphorylation of Akt in CD45+pS6+ cells in salivary glands of WT mice at day 15 pc. (E) Graphs showing phosphatidylinositol (3,4,5)-trisphosphate (PIP3)/phosphatidylinositol (4,5)-biphosphate (PIP2) ratio in salivary glands of mice treated with seletalisib versus vehicle control to demonstrate effect of the compound directly in the salivary glands. Results represented as mean±SD of three independent experiments with five mice per group; **p<0.01, unpaired t-test. (F) Histogram showing pS6 expression levels within the CD45+ cells in day 15 pc salivary glands of mice treated with seletalisib as compared with the vehicle-treated mice. Isotype control also shown. The mice were treated with seletalisib or vehicle from day 12 pc onwards. Data is representative of experiments with three mice in each group.

Journal: Annals of the Rheumatic Diseases

Article Title: Phosphatidylinositol 3-kinase delta pathway: a novel therapeutic target for Sjögren’s syndrome

doi: 10.1136/annrheumdis-2017-212619

Figure Lengend Snippet: (A) Quantitative real-time PCR analysis of PI3KCD transcripts isolated cells from salivary glands of cannulated mice at day 15 postcannulation (pc). B cells (black bar), T cells (dark grey bar), plasma cells (red bar), macrophages (blue) and dendritic cells (light grey bar), CD45− cells (light yellow bars). Results represented as mean±SD from five mice; *p<0.5, ***p<0.001, one-way analysis of variance. (B) Pie chart showing distribution of different leucocyte populations within CD45+ phosphorylated ribosomal protein S6 (pS6+) cells present in salivary glands of wild-type (WT) mice at day 15 pc (C) viSNE plots of flow cytometry of day 15 pc salivary gland CD45+pS6+ cells. Colour indicates cell expression level of labelled marker. Data is representative of two independent experiments with five mice. (D) Histogram showing phosphorylation of Akt in CD45+pS6+ cells in salivary glands of WT mice at day 15 pc. (E) Graphs showing phosphatidylinositol (3,4,5)-trisphosphate (PIP3)/phosphatidylinositol (4,5)-biphosphate (PIP2) ratio in salivary glands of mice treated with seletalisib versus vehicle control to demonstrate effect of the compound directly in the salivary glands. Results represented as mean±SD of three independent experiments with five mice per group; **p<0.01, unpaired t-test. (F) Histogram showing pS6 expression levels within the CD45+ cells in day 15 pc salivary glands of mice treated with seletalisib as compared with the vehicle-treated mice. Isotype control also shown. The mice were treated with seletalisib or vehicle from day 12 pc onwards. Data is representative of experiments with three mice in each group.

Techniques: Real-time Polymerase Chain Reaction, Isolation, Clinical Proteomics, Flow Cytometry, Expressing, Marker, Phospho-proteomics, Control

Fig. 6. Endothelial Jagged1 induces T effector cells via the Notch1-mTORC1 pathway. HMVECs were preconditioned with the indicated plasma, loaded with anti-CD3 antibody, and overlaid with CD4 T cells, as in Fig. 4A. Intracellular expression of pS6, T-bet, or RORgt was analyzed by flow cytometry. All data are means ± SEM. (A and B) pS6 expression in CD4 T cells was measured after 24 hours. Representative histograms and collective MFIs are from three to six independent experiments. (C and D) Effect of the Notch1 signaling inhibitor DAPT (10 mM) or vehicle on the induction of pS6. Data are from six experiments. (E to H) Effect of the mTORC1 inhibitor rapamycin (RAPA; 50 nM) or the AKT inhibitor VIII (AKT Inh; 5 mM) on the induction of T-bet+ and RORgt+ CD4 T cells quantified after 4 days. Data are from five to six experiments. (I) Healthy CD4 T cells were transfected with Raptor siRNA or control siRNA and analyzed for RPTOR mRNA expression 12 hours later by quantitative PCR (qPCR). Data are from five independent experiments. (J) CD4 T cells from healthy donors were transfected with Raptor siRNA or control siRNA, respectively, and cocultured with pretreated ECs, as described above. Data are from six experiments. (K and L) Expression of pS6 in CD4 T cells was measured in freshly isolated PBMCs from GCA patients and healthy donors. Representative histograms and data are from five control-patient pairs. (M to O) Expression of Notch1, pS6, T-bet, and RORgt in CD4 T cells was determined by multiparametric flow cytometry in freshly isolated PBMCs of GCA patients. Each dot represents an individual patient.

Journal: Science translational medicine

Article Title: The microvascular niche instructs T cells in large vessel vasculitis via the VEGF-Jagged1-Notch pathway.

doi: 10.1126/scitranslmed.aal3322

Figure Lengend Snippet: Fig. 6. Endothelial Jagged1 induces T effector cells via the Notch1-mTORC1 pathway. HMVECs were preconditioned with the indicated plasma, loaded with anti-CD3 antibody, and overlaid with CD4 T cells, as in Fig. 4A. Intracellular expression of pS6, T-bet, or RORgt was analyzed by flow cytometry. All data are means ± SEM. (A and B) pS6 expression in CD4 T cells was measured after 24 hours. Representative histograms and collective MFIs are from three to six independent experiments. (C and D) Effect of the Notch1 signaling inhibitor DAPT (10 mM) or vehicle on the induction of pS6. Data are from six experiments. (E to H) Effect of the mTORC1 inhibitor rapamycin (RAPA; 50 nM) or the AKT inhibitor VIII (AKT Inh; 5 mM) on the induction of T-bet+ and RORgt+ CD4 T cells quantified after 4 days. Data are from five to six experiments. (I) Healthy CD4 T cells were transfected with Raptor siRNA or control siRNA and analyzed for RPTOR mRNA expression 12 hours later by quantitative PCR (qPCR). Data are from five independent experiments. (J) CD4 T cells from healthy donors were transfected with Raptor siRNA or control siRNA, respectively, and cocultured with pretreated ECs, as described above. Data are from six experiments. (K and L) Expression of pS6 in CD4 T cells was measured in freshly isolated PBMCs from GCA patients and healthy donors. Representative histograms and data are from five control-patient pairs. (M to O) Expression of Notch1, pS6, T-bet, and RORgt in CD4 T cells was determined by multiparametric flow cytometry in freshly isolated PBMCs of GCA patients. Each dot represents an individual patient.

Article Snippet: Multiparametric flow cytometry panels were assembled with antibodies to Notch1, CD45RA, CD25, HES1, T-bet, RORgt, and pS6, combined with anti-CD3 and anti-CD4 antibodies as follows: APC-Cy7 (allophycocyanin-Cy7) anti-human CD3 antibody (clone HIT3a, BioLegend), FITC (fluorescein isothiocyanate) or PE-Cy7 (phycoerythrinCy7) anti-human CD4 antibody (clone RPA-T4, BioLegend), APC or APC-Cy7 anti-human CD45RA antibody (clone HI100, BioLegend), PE anti-humanCD25antibody (cloneM-A251, BioLegend),APCorPE antihuman Notch1 antibody (clone MHN1-519, BioLegend), purified antihuman HES1 antibody (clone 4H1HES, eBioscience) plus PE-Cy7 anti-mouse IgG1 antibody (clone M1-14D12, eBioscience), PE or PE-Cy7 anti-human T-bet antibody (clone eBio4B10, eBioscience), APC anti-human RORgt antibody (clone AFKJS-9, eBioscience), Parameters Patients with GCA Patients with RA Number of patients 80 54 Age (years) (mean ± SD) 69.63 ± 7.95 53.16 ± 16.35 Female 77.5% 79.63% Headaches 70% Eye involvement 31.25% Jaw claudication 26.25% Polymyalgia rheumatica 60% Aortic/large vessel involvement 43.75% Prednisone (mg/day) (mean ± SD) 7.49 ± 11.29 Disease duration (months) (mean ± SD) 14.36 ± 12.40 14.88 ± 11.48 ESR (mm/hour) (mean ± SD) 51.43 ± 35.39 24.63 ± 19.53 CRP (mg/dl) (mean ± SD) 6.25 ± 7.48 1.72 ± 4.47 Untreated patients 32.5% 16.67% 12 of 15 SC I ENCE TRANS LAT IONAL MED I C I N E | R E S EARCH ART I C L E D ow nloaded from https://w w w .science.org on January 23, 2024 and PE anti-human pS6 antibody (cloneD57.2.2E, Cell Signaling Technology).

Techniques: Clinical Proteomics, Expressing, Flow Cytometry, Transfection, Control, Real-time Polymerase Chain Reaction, Isolation

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