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Monensin Solution 1 000X

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Monensin Solution 1 000X Apps ICFC Size 1 ml

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BioLegend monensin

Induction of differentiation, inhibition of cell growth, and resistance to NK cell-mediated cytotoxicity in OSCSCs induced by IL-2 + anti-CD16 mAb-treated and paraformaldehyde-fixed NK cells is mediated by the combination of IFN-γ and TNF-α and not each cytokine alone . Highly purified NK cells were left untreated or treated with the combination of IL-2 (1000 units/ml) and anti-CD16 mAb (3 μg/ml) for 24 h, after which the NK cells were fixed with 2% paraformaldehyde and added to OSCSCs (0.75:1 NK:OSCSC ratio) in the presence and absence of anti-TNF-α (1:100) and/or anti-IFN-γ (1:100) or isotype control antibodies for a period of 5 days. The media containing the fixed NK cells were removed and extensively washed from each treated OSCSCs and the cytotoxicity against OSCSCs were assessed using freshly isolated untreated NK cells or those treated with IL-2 using a standard 4 h 51 Cr release assay. The complete removal of fixed NK cells from OSCSCs was determined by microscopy. Percent cytotoxicity was obtained at different effector to target ratio, and the lytic units 30/10 6 cells were determined using inverse number of NK cells required to lyse 30% of the tumor cells × 100 (A) . The lack of cytotoxic function of 2% paraformaldehyde-fixed untreated and IL-2-treated NK cells were assessed against OSCSCs and OSCCs. NK cells were left untreated or treated with IL-2 for 24 h before they were fixed with 2% paraformaldehyde (B) . NK cells were purified from healthy donor and left untreated or treated with IL-2 (1000 units/ml) and the combination of IL-2 (1000 units/ml) and anti-CD16 mAb (3 μg/ml). After an overnight treatment, NK cells were fixed with freshly prepared 2% paraformaldehyde and washed twice with 1× PBS. After 24 h post fixation, supernatants were then collected and the levels of IFN-γ were measured with specific ELISA (C) . NK cells purified from healthy donors were left untreated or treated with a combination of IL-2 (1000 units/ml) and anti-CD16 mAb (3 μg/ml) for 24 h, after which the NK cells were washed extensively and stained with PE-conjugated anti-TNF-α mAb followed by flow cytometric analysis. IFN-γ expression was assessed using purified mouse anti-human IFN-γ antibody followed by PE-conjugated goat anti-mouse IgG. Isotype control antibodies were used as controls. The numbers on the right hand corner are percentage positive for each histogram. The histogram overlay for the anti-TNF-α or anti-IFN-γ stained untreated (left) and IL-2 + anti-CD16 mAb-treated NK cells (right) are also shown in this figure. (D) The surface expression of CD54, CD44, B7H1, and MHC class I on untreated and fixed NK-treated OSCSCs as described above were assessed after PE-conjugated antibody staining followed by flow cytometric analysis. Isotype control antibodies were used as controls. The numbers on the right hand corner are the percentages and the mean channel fluorescence intensities for each histogram (E) . At the end of the incubat ion of OSCSCs with fixed NK cells, OSCSCs which were remained attached to the plate were collected, and the number of cells (F) and their viability (G) were assessed using microscopy, and propidium iodide staining followed by flow cytometric analysis, respectively. Highly purified NK cells were left untreated or treated with the combination of IL-2 (1000 units/ml) and anti-CD16 mAb (3 μg/ml) in the presence and absence of <t>monensin</t> (1:1300) for 24 h, after which each sample of NK cells were extensively washed and fixed with 2% paraformaldehyde and added to OSCSCs (0.75:1 NK:OSCSC) for a period of 5 days. Inhibition of IFN-γ release in monensin-treated NK cells before fixation with 2% paraformaldehyde were assessed by ELISA (H) . The results were compared to OSCSCs in the absence of fixed NK cells. The media containing the fixed NK cells were removed and extensively washed from each treated OSCSCs and the cytotoxicity against each condition of OSCSCs were assessed using freshly isolated untreated NK cells (I) , those treated with IL-2 (J) or treated with the combination of IL-2 + anti-CD16 mAb (K) using a standard 4 h 51 Cr release assay. The complete removal of fixed NK cells from OSCSCs was determined by microscopy. Differentiated OSCCs were used as control for the differentiation of OSCSCs. Percent cytotoxicity was obtained at different effector to target ratio, and the lytic units 30/10 6 cells were determined using inverse number of NK cells required to lyse 30% of the tumor cells × 100. The surface expression of CD54, CD44, and MHC class I on untreated and fixed NK-treated OSCSCs as described above were assessed after PE-conjugated antibody staining followed by flow cytometric analysis. Isotype control antibodies were used as controls. The numbers on the right hand corner are the percentages and the mean channel fluorescence intensities for each histogram (L) . At the end of the incubation of OSCSCs with fixed NK cells, the media containing the fixed NK samples were removed from OSCSCs and the viability of the cells were assessed using propidium iodide staining followed by flow cytometric analysis (M) . One of minimum three representative experiments is shown in each of (A–M) .

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1) Product Images from "Induction of Split Anergy Conditions Natural Killer Cells to Promote Differentiation of Stem Cells through Cell-Cell Contact and Secreted Factors"

Article Title: Induction of Split Anergy Conditions Natural Killer Cells to Promote Differentiation of Stem Cells through Cell-Cell Contact and Secreted Factors

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2014.00269

Figure Legend Snippet: Induction of differentiation, inhibition of cell growth, and resistance to NK cell-mediated cytotoxicity in OSCSCs induced by IL-2 + anti-CD16 mAb-treated and paraformaldehyde-fixed NK cells is mediated by the combination of IFN-γ and TNF-α and not each cytokine alone . Highly purified NK cells were left untreated or treated with the combination of IL-2 (1000 units/ml) and anti-CD16 mAb (3 μg/ml) for 24 h, after which the NK cells were fixed with 2% paraformaldehyde and added to OSCSCs (0.75:1 NK:OSCSC ratio) in the presence and absence of anti-TNF-α (1:100) and/or anti-IFN-γ (1:100) or isotype control antibodies for a period of 5 days. The media containing the fixed NK cells were removed and extensively washed from each treated OSCSCs and the cytotoxicity against OSCSCs were assessed using freshly isolated untreated NK cells or those treated with IL-2 using a standard 4 h 51 Cr release assay. The complete removal of fixed NK cells from OSCSCs was determined by microscopy. Percent cytotoxicity was obtained at different effector to target ratio, and the lytic units 30/10 6 cells were determined using inverse number of NK cells required to lyse 30% of the tumor cells × 100 (A) . The lack of cytotoxic function of 2% paraformaldehyde-fixed untreated and IL-2-treated NK cells were assessed against OSCSCs and OSCCs. NK cells were left untreated or treated with IL-2 for 24 h before they were fixed with 2% paraformaldehyde (B) . NK cells were purified from healthy donor and left untreated or treated with IL-2 (1000 units/ml) and the combination of IL-2 (1000 units/ml) and anti-CD16 mAb (3 μg/ml). After an overnight treatment, NK cells were fixed with freshly prepared 2% paraformaldehyde and washed twice with 1× PBS. After 24 h post fixation, supernatants were then collected and the levels of IFN-γ were measured with specific ELISA (C) . NK cells purified from healthy donors were left untreated or treated with a combination of IL-2 (1000 units/ml) and anti-CD16 mAb (3 μg/ml) for 24 h, after which the NK cells were washed extensively and stained with PE-conjugated anti-TNF-α mAb followed by flow cytometric analysis. IFN-γ expression was assessed using purified mouse anti-human IFN-γ antibody followed by PE-conjugated goat anti-mouse IgG. Isotype control antibodies were used as controls. The numbers on the right hand corner are percentage positive for each histogram. The histogram overlay for the anti-TNF-α or anti-IFN-γ stained untreated (left) and IL-2 + anti-CD16 mAb-treated NK cells (right) are also shown in this figure. (D) The surface expression of CD54, CD44, B7H1, and MHC class I on untreated and fixed NK-treated OSCSCs as described above were assessed after PE-conjugated antibody staining followed by flow cytometric analysis. Isotype control antibodies were used as controls. The numbers on the right hand corner are the percentages and the mean channel fluorescence intensities for each histogram (E) . At the end of the incubat ion of OSCSCs with fixed NK cells, OSCSCs which were remained attached to the plate were collected, and the number of cells (F) and their viability (G) were assessed using microscopy, and propidium iodide staining followed by flow cytometric analysis, respectively. Highly purified NK cells were left untreated or treated with the combination of IL-2 (1000 units/ml) and anti-CD16 mAb (3 μg/ml) in the presence and absence of monensin (1:1300) for 24 h, after which each sample of NK cells were extensively washed and fixed with 2% paraformaldehyde and added to OSCSCs (0.75:1 NK:OSCSC) for a period of 5 days. Inhibition of IFN-γ release in monensin-treated NK cells before fixation with 2% paraformaldehyde were assessed by ELISA (H) . The results were compared to OSCSCs in the absence of fixed NK cells. The media containing the fixed NK cells were removed and extensively washed from each treated OSCSCs and the cytotoxicity against each condition of OSCSCs were assessed using freshly isolated untreated NK cells (I) , those treated with IL-2 (J) or treated with the combination of IL-2 + anti-CD16 mAb (K) using a standard 4 h 51 Cr release assay. The complete removal of fixed NK cells from OSCSCs was determined by microscopy. Differentiated OSCCs were used as control for the differentiation of OSCSCs. Percent cytotoxicity was obtained at different effector to target ratio, and the lytic units 30/10 6 cells were determined using inverse number of NK cells required to lyse 30% of the tumor cells × 100. The surface expression of CD54, CD44, and MHC class I on untreated and fixed NK-treated OSCSCs as described above were assessed after PE-conjugated antibody staining followed by flow cytometric analysis. Isotype control antibodies were used as controls. The numbers on the right hand corner are the percentages and the mean channel fluorescence intensities for each histogram (L) . At the end of the incubation of OSCSCs with fixed NK cells, the media containing the fixed NK samples were removed from OSCSCs and the viability of the cells were assessed using propidium iodide staining followed by flow cytometric analysis (M) . One of minimum three representative experiments is shown in each of (A–M) .

Techniques Used: Inhibition, Purification, Isolation, Release Assay, Microscopy, Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry, Expressing, Fluorescence, Incubation

2) Product Images from "Human intestinal tissue-resident memory T cells comprise transcriptionally and functionally distinct subsets"

Article Title: Human intestinal tissue-resident memory T cells comprise transcriptionally and functionally distinct subsets

Journal: Cell Reports

doi: 10.1016/j.celrep.2020.108661

CD103 + and CD103 − CD4 + T cells display analogous phenotypic and functional differences to their CD8 + counterparts (A) MFI (CD161, CD7, CD127, β2-integrin, granzyme K, and KLRG1) or percentage positive (Ki-67) of CD4 + T cells, categorized by CD69 and CD103 expression, in small intestinal biopsies from healthy control subjects (n = 10). MFI represented by bars. Connecting lines represent populations from the same subject. Blue, CD69 − CD103 − cells; red, CD69 + CD103 − cells; black, CD69 + CD103 + cells. (B) MFI of β2-integrin on recipient-derived CD4 + T cells, categorized by CD69 and CD103 expression and time after transplant, in intestinal transplant grafts (n = 21; 12 subjects). MFI represented by bars. (C–H) Cytokine production by small intestinal CD4 + T cells. Representative histograms of expression, and group summaries of proportion of CD4 + T cells expressing IL-17A (C), TNF-α (D), IFN-γ (E), IL-2 (F), CCL4 (G), and IL-10 (H) after 4 h stimulation with PMA and ionomycin in the presence of brefeldin A and monensin, categorized by CD69 and CD103 expression, in small intestinal biopsies from healthy control subjects (n = 10). (I) Mean proportion of CD4 + T cells expressing 0, 1, 2, 3, 4, 5, or 6 of the cytokines or chemokines IL-17A, TNF-α, IFN-γ, CCL4, IL-2, and IL-10, categorized by CD69 and CD103 co-expression, from small intestinal biopsies from healthy control subjects (n = 10). Statistical analysis performed with one-way ANOVA with Tukey’s multiple-comparison test. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001.

Figure Legend Snippet: CD103 + and CD103 − CD4 + T cells display analogous phenotypic and functional differences to their CD8 + counterparts (A) MFI (CD161, CD7, CD127, β2-integrin, granzyme K, and KLRG1) or percentage positive (Ki-67) of CD4 + T cells, categorized by CD69 and CD103 expression, in small intestinal biopsies from healthy control subjects (n = 10). MFI represented by bars. Connecting lines represent populations from the same subject. Blue, CD69 − CD103 − cells; red, CD69 + CD103 − cells; black, CD69 + CD103 + cells. (B) MFI of β2-integrin on recipient-derived CD4 + T cells, categorized by CD69 and CD103 expression and time after transplant, in intestinal transplant grafts (n = 21; 12 subjects). MFI represented by bars. (C–H) Cytokine production by small intestinal CD4 + T cells. Representative histograms of expression, and group summaries of proportion of CD4 + T cells expressing IL-17A (C), TNF-α (D), IFN-γ (E), IL-2 (F), CCL4 (G), and IL-10 (H) after 4 h stimulation with PMA and ionomycin in the presence of brefeldin A and monensin, categorized by CD69 and CD103 expression, in small intestinal biopsies from healthy control subjects (n = 10). (I) Mean proportion of CD4 + T cells expressing 0, 1, 2, 3, 4, 5, or 6 of the cytokines or chemokines IL-17A, TNF-α, IFN-γ, CCL4, IL-2, and IL-10, categorized by CD69 and CD103 co-expression, from small intestinal biopsies from healthy control subjects (n = 10). Statistical analysis performed with one-way ANOVA with Tukey’s multiple-comparison test. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001.

Techniques Used: Functional Assay, Expressing, Derivative Assay

CD103 + CD8 + intestinal T cells demonstrate greater capacity for cytokine production (A–D) Cytokine production by small intestinal CD8 + T cells. Representative histograms of expression, and group summaries of proportion of CD8 + T cells expressing TNF-α (A), IFN-γ (B), CCL4 (C), and IL-2 (D) after 4 h stimulation with PMA and ionomycin in the presence of brefeldin A and monensin, categorized by CD69 and CD103 expression, in small intestinal biopsies from healthy control subjects (n = 10). (E) Mean proportion of CD8 + T cells expressing 0, 1, 2, 3, or 4 of the cytokines/chemokines TNF-α, IFN-γ, CCL4, and IL-2, categorized by CD69 and CD103 co-expression, from small intestinal biopsies from healthy control subjects (n = 10). (F) Mean percentage (± SEM) of CD8 + T cells co-expressing TNF-α, IFN-γ, CCL4, and/or IL-2 after PMA and ionomycin stimulation as described, categorized by CD69 and CD103 expression. Statistical analysis performed with one-way ANOVA with Tukey’s multiple-comparison test. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001.

Figure Legend Snippet: CD103 + CD8 + intestinal T cells demonstrate greater capacity for cytokine production (A–D) Cytokine production by small intestinal CD8 + T cells. Representative histograms of expression, and group summaries of proportion of CD8 + T cells expressing TNF-α (A), IFN-γ (B), CCL4 (C), and IL-2 (D) after 4 h stimulation with PMA and ionomycin in the presence of brefeldin A and monensin, categorized by CD69 and CD103 expression, in small intestinal biopsies from healthy control subjects (n = 10). (E) Mean proportion of CD8 + T cells expressing 0, 1, 2, 3, or 4 of the cytokines/chemokines TNF-α, IFN-γ, CCL4, and IL-2, categorized by CD69 and CD103 co-expression, from small intestinal biopsies from healthy control subjects (n = 10). (F) Mean percentage (± SEM) of CD8 + T cells co-expressing TNF-α, IFN-γ, CCL4, and/or IL-2 after PMA and ionomycin stimulation as described, categorized by CD69 and CD103 expression. Statistical analysis performed with one-way ANOVA with Tukey’s multiple-comparison test. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001.

Techniques Used: Expressing

3) Product Images from "STAT5 promotes accessibility and is required for BATF-mediated plasticity at the Il9 locus"

Article Title: STAT5 promotes accessibility and is required for BATF-mediated plasticity at the Il9 locus

Journal: Nature Communications

doi: 10.1038/s41467-020-18648-6

Cooperation between STAT5 and BATF in the plasticity of the Il9 locus. Naive CD4+ T cells were isolated from the spleen and differentiated into Th lineage for 5 days. Retrovirus expressing caSTAT5 (tagged with hNGFR), BATF (tagged with Thy1.1), or IRF4 (tagged with GFP) was transduced on day 1. For cytokine production analysis, cells were stimulated with PMA/Ionomycin for 5 h and monensin was added for the last 2 h. a Venn diagram of overlap between BATF and STAT5b target genes in Th9 cells. b , c Flow cytometric analysis of cytokine expression in Th17 cells co-transduced with caSTAT5 and BATF retrovirus. d Flow cytometric analysis of IL-9 expression in Th17 cells co-transduced with caSTAT5 and IRF4 retrovirus; cells were analyzed on day 5. e , f Flow cytometric analysis of cytokine expression in WT or Batf ΔZ/ΔZ Th9 cells co-transduced with caSTAT5 and BATF retrovirus; cells were analyzed on day 5. g , h Flow cytometric analysis of cytokine expression in WT or Batf ΔZ/ΔZ Th17 cells co-transduced with caSTAT5 and BATF retrovirus; cells were analyzed on day 5. i Flow cytometric analysis of IL-9 expression in caSTAT5 lone or caSTAT5 and BATF retrovirus co-transduced Th0, Th1, Th2, and Treg cells, cells were analyzed on day 5. Date are mean ± SEM of three mice per experiment and representative of two independent experiments. Two-way ANOVA with Sidak’s multiple comparison test was used to generate p -values. ns: no significant, p > 0.05. See also Supplementary Fig. 4 .

Figure Legend Snippet: Cooperation between STAT5 and BATF in the plasticity of the Il9 locus. Naive CD4+ T cells were isolated from the spleen and differentiated into Th lineage for 5 days. Retrovirus expressing caSTAT5 (tagged with hNGFR), BATF (tagged with Thy1.1), or IRF4 (tagged with GFP) was transduced on day 1. For cytokine production analysis, cells were stimulated with PMA/Ionomycin for 5 h and monensin was added for the last 2 h. a Venn diagram of overlap between BATF and STAT5b target genes in Th9 cells. b , c Flow cytometric analysis of cytokine expression in Th17 cells co-transduced with caSTAT5 and BATF retrovirus. d Flow cytometric analysis of IL-9 expression in Th17 cells co-transduced with caSTAT5 and IRF4 retrovirus; cells were analyzed on day 5. e , f Flow cytometric analysis of cytokine expression in WT or Batf ΔZ/ΔZ Th9 cells co-transduced with caSTAT5 and BATF retrovirus; cells were analyzed on day 5. g , h Flow cytometric analysis of cytokine expression in WT or Batf ΔZ/ΔZ Th17 cells co-transduced with caSTAT5 and BATF retrovirus; cells were analyzed on day 5. i Flow cytometric analysis of IL-9 expression in caSTAT5 lone or caSTAT5 and BATF retrovirus co-transduced Th0, Th1, Th2, and Treg cells, cells were analyzed on day 5. Date are mean ± SEM of three mice per experiment and representative of two independent experiments. Two-way ANOVA with Sidak’s multiple comparison test was used to generate p -values. ns: no significant, p > 0.05. See also Supplementary Fig. 4 .

Techniques Used: Isolation, Expressing, Transduction, Mouse Assay

Accessibility is required for BATF to activate Il9 . Naive CD4+ T cells were isolated from spleen and differentiated into Th9 and Th17 cells for 5 days ( a – c ) or 6 days ( d – h ). dCas9/dCas9-VP64- and gRNA-expressing retrovirus were transduced on day 1; BATF, IRF4, and control vector was transduced on day 4. ChIP assay and chromatin accessibility assay were performed on day 5 or day 6. For cytokine production analysis, Th9 or Th17 cells were stimulated with phorbol 12-myristate 13-acetate (PMA)/Ionomycin for 5 h and monensin was added for the last 2 h. a Flow cytometry analysis of IL-9 production in WT, Batf ΔZ/ΔZ , and Stat6 −/− Th9 cells on day 5 ( n = 3 per group). b Binding of RNA polymerase II on the Il9 promoter as determined by ChIP-qPCR on day 5 ( n = 3 per group). c Chromatin accessibility analysis of Il9 locus in WT, Batf ΔZ/ΔZ , and Stat6 −/− Th9 cells on day 5 ( n = 3 per group). d Th17 cells transduced with control vectors or dCas9-VP64 and gRNA-CNS1 were sorted and chromatin accessibility of Il9 locus was detected on day 6 ( n = 4 for control vector group, n = 3 for dCas9-VP64 gRNA-CNS1 group). e Th17 cells were transduced with dCas9-VP64 and gRNA-CNS1, followed by BATF or control vector transduction. Transduced cells were sorted on day 6 and chromatin accessibility of the Il9 locus was assessed ( n = 4 for dCas9 gRNA-CNS1 group, n = 3 for dCas9-VP64 gRNA-CNS1 group). f Flow cytometry analysis of IL-9 expression in Th17 cells transduced with retrovirus expressing gRNA-CNS1, dCas9-VP64, BATF, or control vectors on day 6 ( n = 3 per group). g Flow cytometry analysis of IL-9 expression in Th17 cells transduced with retrovirus expressing gRNA-CNS-25, dCas9-VP64, BATF, or control vectors on day 6 ( n = 3 per group). h Flow cytometry analysis of IL-9 expression in Th17 cells transduced with retrovirus expressing gRNA-CNS-25, dCas9-VP64, IRF4, or control vectors on day 6 ( n = 3 per group). Histograms are gated on transduced CD4+ T cells. Data are mean ± SEM. a , b One-way ANOVA with a Dunnett’s multiple comparison test was used to generate p -values for multiple comparisons. c Unpaired two-tailed Student’s t -test was used for comparison. f , g Two-way ANOVA with Sidak’s multiple comparisons was used to generate p -values. See also Supplementary Fig. 2 .

Figure Legend Snippet: Accessibility is required for BATF to activate Il9 . Naive CD4+ T cells were isolated from spleen and differentiated into Th9 and Th17 cells for 5 days ( a – c ) or 6 days ( d – h ). dCas9/dCas9-VP64- and gRNA-expressing retrovirus were transduced on day 1; BATF, IRF4, and control vector was transduced on day 4. ChIP assay and chromatin accessibility assay were performed on day 5 or day 6. For cytokine production analysis, Th9 or Th17 cells were stimulated with phorbol 12-myristate 13-acetate (PMA)/Ionomycin for 5 h and monensin was added for the last 2 h. a Flow cytometry analysis of IL-9 production in WT, Batf ΔZ/ΔZ , and Stat6 −/− Th9 cells on day 5 ( n = 3 per group). b Binding of RNA polymerase II on the Il9 promoter as determined by ChIP-qPCR on day 5 ( n = 3 per group). c Chromatin accessibility analysis of Il9 locus in WT, Batf ΔZ/ΔZ , and Stat6 −/− Th9 cells on day 5 ( n = 3 per group). d Th17 cells transduced with control vectors or dCas9-VP64 and gRNA-CNS1 were sorted and chromatin accessibility of Il9 locus was detected on day 6 ( n = 4 for control vector group, n = 3 for dCas9-VP64 gRNA-CNS1 group). e Th17 cells were transduced with dCas9-VP64 and gRNA-CNS1, followed by BATF or control vector transduction. Transduced cells were sorted on day 6 and chromatin accessibility of the Il9 locus was assessed ( n = 4 for dCas9 gRNA-CNS1 group, n = 3 for dCas9-VP64 gRNA-CNS1 group). f Flow cytometry analysis of IL-9 expression in Th17 cells transduced with retrovirus expressing gRNA-CNS1, dCas9-VP64, BATF, or control vectors on day 6 ( n = 3 per group). g Flow cytometry analysis of IL-9 expression in Th17 cells transduced with retrovirus expressing gRNA-CNS-25, dCas9-VP64, BATF, or control vectors on day 6 ( n = 3 per group). h Flow cytometry analysis of IL-9 expression in Th17 cells transduced with retrovirus expressing gRNA-CNS-25, dCas9-VP64, IRF4, or control vectors on day 6 ( n = 3 per group). Histograms are gated on transduced CD4+ T cells. Data are mean ± SEM. a , b One-way ANOVA with a Dunnett’s multiple comparison test was used to generate p -values for multiple comparisons. c Unpaired two-tailed Student’s t -test was used for comparison. f , g Two-way ANOVA with Sidak’s multiple comparisons was used to generate p -values. See also Supplementary Fig. 2 .

Techniques Used: Isolation, Expressing, Plasmid Preparation, Chromatin Immunoprecipitation, Flow Cytometry, Binding Assay, Real-time Polymerase Chain Reaction, Transduction, Two Tailed Test

STAT5 promotes the accessibility of IL9 gene for BATF binding in human Th9 cells. Naive CD4+ cells were isolated from human peripheral blood mononuclear cells; cells were differentiated to Th9 cells ( a – i ). ChIP assay and chromatin accessibility assay were performed on day 5. STAT5 inhibitor was added to the culture on day 1. For cytokine production analysis, cells were stimulated with PMA/Ionomycin for 5 h and monensin was added for the last 2 h. For j – m , peripheral blood mononuclear cells or sorted T cells from pediatric asthmatic or non-asthmatic dermatitis patients were analyzed as described. a , b Kinetic analysis of pSTAT5 detection from naive human CD4+ cells to D5 Th9 culture ( n = 3). c Kinetic analysis of IL-9 expression from D0 to D5 Th9 culture ( n = 4 for D0 to D2 groups, n = 3 for D3 to D5 groups). d , e Kinetic analysis of IL9 gene accessibility from naive human CD4+ cells to D5 Th9 culture ( n = 3). f IL-9 expression in Th9 cells transduced with Scr-shRNA or STAT5b-shRNA lentivirus, cells were analyzed on day 5 ( n = 4). g Th9 cells treated with DMSO or STAT5 inhibitor on day 1, IL-9 and pSTAT5 were analyzed on day 5 ( n = 4). h Chromatin accessibility analysis of IL9 gene locus in Th9 cells treated with DMSO or STAT5 inhibitor; cells were analyzed on day 5 ( n = 3). i The binding of BATF at the IL9 gene locus in Th9 cells treated with DMSO or STAT5 inhibitor ( n = 3). j ELISA analysis of IL-9 expression in the peripheral blood mononuclear cells from patient samples stimulated with anti-CD3 for 12 h ( n = 4 for non-asthma group, n = 6 for asthma group). k Flow cytometric analysis of CCR4+CD4+ cells on peripheral blood mononuclear cells from non-asthma or asthma patients ( n = 4 for non-asthma group, n = 6 for asthma group). l Flow cytometric analysis of pSTAT5 expression cells in peripheral blood mononuclear cells from non-asthma or asthma patients stimulated with 100 U/ml IL-2 for 10 min ( n = 4 for non-asthma group, n = 7 for asthma group). m Chromatin accessibility analysis of the IL9 gene locus in CCR4+CD4+ cells sorted from patient peripheral blood mononuclear cells ( n = 4 for non-asthma group, n = 3 for asthma group). Date are mean ± SEM. One-way ANOVA with a Dunnett’s multiple comparison test was used to generate p -values for all multiple comparisons in b – e . An unpaired two-tailed Student t-test was used for comparisons in f – m .

Figure Legend Snippet: STAT5 promotes the accessibility of IL9 gene for BATF binding in human Th9 cells. Naive CD4+ cells were isolated from human peripheral blood mononuclear cells; cells were differentiated to Th9 cells ( a – i ). ChIP assay and chromatin accessibility assay were performed on day 5. STAT5 inhibitor was added to the culture on day 1. For cytokine production analysis, cells were stimulated with PMA/Ionomycin for 5 h and monensin was added for the last 2 h. For j – m , peripheral blood mononuclear cells or sorted T cells from pediatric asthmatic or non-asthmatic dermatitis patients were analyzed as described. a , b Kinetic analysis of pSTAT5 detection from naive human CD4+ cells to D5 Th9 culture ( n = 3). c Kinetic analysis of IL-9 expression from D0 to D5 Th9 culture ( n = 4 for D0 to D2 groups, n = 3 for D3 to D5 groups). d , e Kinetic analysis of IL9 gene accessibility from naive human CD4+ cells to D5 Th9 culture ( n = 3). f IL-9 expression in Th9 cells transduced with Scr-shRNA or STAT5b-shRNA lentivirus, cells were analyzed on day 5 ( n = 4). g Th9 cells treated with DMSO or STAT5 inhibitor on day 1, IL-9 and pSTAT5 were analyzed on day 5 ( n = 4). h Chromatin accessibility analysis of IL9 gene locus in Th9 cells treated with DMSO or STAT5 inhibitor; cells were analyzed on day 5 ( n = 3). i The binding of BATF at the IL9 gene locus in Th9 cells treated with DMSO or STAT5 inhibitor ( n = 3). j ELISA analysis of IL-9 expression in the peripheral blood mononuclear cells from patient samples stimulated with anti-CD3 for 12 h ( n = 4 for non-asthma group, n = 6 for asthma group). k Flow cytometric analysis of CCR4+CD4+ cells on peripheral blood mononuclear cells from non-asthma or asthma patients ( n = 4 for non-asthma group, n = 6 for asthma group). l Flow cytometric analysis of pSTAT5 expression cells in peripheral blood mononuclear cells from non-asthma or asthma patients stimulated with 100 U/ml IL-2 for 10 min ( n = 4 for non-asthma group, n = 7 for asthma group). m Chromatin accessibility analysis of the IL9 gene locus in CCR4+CD4+ cells sorted from patient peripheral blood mononuclear cells ( n = 4 for non-asthma group, n = 3 for asthma group). Date are mean ± SEM. One-way ANOVA with a Dunnett’s multiple comparison test was used to generate p -values for all multiple comparisons in b – e . An unpaired two-tailed Student t-test was used for comparisons in f – m .

Techniques Used: Binding Assay, Isolation, Chromatin Immunoprecipitation, Expressing, Transduction, shRNA, Enzyme-linked Immunosorbent Assay, Two Tailed Test

STAT5 regulates Il9 chromatin accessibility. Naive CD4+ T cells were isolated from the spleen and differentiated into Th9 and Th17 cells for 5 days. ChIP assay and chromatin accessibility assay were performed on day 5. shRNA-expressing retrovirus was transduced on day 1. For cytokine production analysis, cells were stimulated with PMA/Ionomycin for 5 h and monensin was added for the last 2 h. a – c Kinetic analysis of chromatin accessibility, transcription factors, and chromatin modification markers binding on Il9 promoter locus in Th9 cells during Th9 differentiation. d FACS analysis of IL-9 and pSTAT5 expression in Th9 cells transduced with control (Scr), STAT5a-specific, or STAT5b-specific shRNA; cells were gated on transduced CD4+ live cells. e Chromatin accessibility analysis of Il9 gene locus in Th9 cells transduced with Scr-shRNA or STAT5a-shRNA retrovirus. f , g H3K27me3 modification and BATF binding at the Il9 gene locus in Th9 cells transduced with Scr-shRNA or STAT5a-shRNA retrovirus on day 5. h , i FACS analysis of IL-9 and IL-17 expression on day 5 in Th17 cells transduced with control vector or caSTAT5 retrovirus; cells were gated on transduced CD4+ live cells. j Chromatin accessibility analysis of Il9 gene locus in Th17 cells that transduced with control vector or caSTAT5 retrovirus; transduced cells were sorted on day 5. k The binding of BATF on Il9 gene locus in Th17 cells that transduced with control vector or caSTAT5 retrovirus; transduced cells were sorted on day 5. Date are mean ± SEM of three mice per experiment and representative of two independent experiments. The p -values for a and b are compared to D0 for chromatin accessibility and STAT5 binding; D1 for other transcription factors and chromatin modification markers binding. One-way ANOVA with a post hoc Tukey’s test was used to generate p -values for all multiple comparisons in b . An unpaired two-tailed Student’s t -test was used for comparisons in a , d , e , h , j , and k . See also Supplementary Fig. 3 .

Figure Legend Snippet: STAT5 regulates Il9 chromatin accessibility. Naive CD4+ T cells were isolated from the spleen and differentiated into Th9 and Th17 cells for 5 days. ChIP assay and chromatin accessibility assay were performed on day 5. shRNA-expressing retrovirus was transduced on day 1. For cytokine production analysis, cells were stimulated with PMA/Ionomycin for 5 h and monensin was added for the last 2 h. a – c Kinetic analysis of chromatin accessibility, transcription factors, and chromatin modification markers binding on Il9 promoter locus in Th9 cells during Th9 differentiation. d FACS analysis of IL-9 and pSTAT5 expression in Th9 cells transduced with control (Scr), STAT5a-specific, or STAT5b-specific shRNA; cells were gated on transduced CD4+ live cells. e Chromatin accessibility analysis of Il9 gene locus in Th9 cells transduced with Scr-shRNA or STAT5a-shRNA retrovirus. f , g H3K27me3 modification and BATF binding at the Il9 gene locus in Th9 cells transduced with Scr-shRNA or STAT5a-shRNA retrovirus on day 5. h , i FACS analysis of IL-9 and IL-17 expression on day 5 in Th17 cells transduced with control vector or caSTAT5 retrovirus; cells were gated on transduced CD4+ live cells. j Chromatin accessibility analysis of Il9 gene locus in Th17 cells that transduced with control vector or caSTAT5 retrovirus; transduced cells were sorted on day 5. k The binding of BATF on Il9 gene locus in Th17 cells that transduced with control vector or caSTAT5 retrovirus; transduced cells were sorted on day 5. Date are mean ± SEM of three mice per experiment and representative of two independent experiments. The p -values for a and b are compared to D0 for chromatin accessibility and STAT5 binding; D1 for other transcription factors and chromatin modification markers binding. One-way ANOVA with a post hoc Tukey’s test was used to generate p -values for all multiple comparisons in b . An unpaired two-tailed Student’s t -test was used for comparisons in a , d , e , h , j , and k . See also Supplementary Fig. 3 .

Techniques Used: Isolation, Chromatin Immunoprecipitation, shRNA, Expressing, Modification, Binding Assay, FACS, Transduction, Plasmid Preparation, Mouse Assay, Two Tailed Test

Incubation:

Article Title: Deletion of F4L (ribonucleotide reductase) in vaccinia virus produces a selective oncolytic virus and promotes anti‐tumor immunity with superior safety in bladder cancer models
Article Snippet: CD107a expression was measured essentially as described by Betts et al ( ). .. BMDCs were added to CD3+ cells, co‐cultured for 1 h in the presence of CD107a antibody, and incubated for another 5 h in the presence of monensin and brefeldin A (BioLegend). .. The cells were fixed and stained with anti‐CD4, anti‐CD8, and secondary to CD107a antibodies and analyzed by flow cytometry.

Article Title: In vivo depletion of CD4+CD25hi regulatory T cells is associated with improved antiviral responses in cats chronically infected with feline immunodeficiency virus
Article Snippet: Anti-CD8α (3.357) was conjugated to PerCP using a kit from Prozyme (San Leandro, CA). .. Intracellular FOXP3 staining was performed with eBioscience FOXP3 staining buffers and FOXP3-PECy7 (FJK-16s; San Diego, CA) according to manufacturer’s protocol, with the exception that cells remain in the permeabilization/wash buffer no longer than 30 min. For intracellular cytokine staining, cells were incubated with 1x monensin (Biolegend, San Diego, CA) for six hours, labeled for surface markers, fixed with 4% paraformaldehyde, permeabilized with BD Cytofix/Cytoperm kit Perm/Wash buffer, and stained with IL-2-PE (MQ1-17H12; BioLegend) and tumor necrosis factor (TNF)-α-APC (6401.1111; BD). .. Flow cytometric analysis was performed using an LSR II flow cytometer and FacsDIVA software (BD).

Article Title: Human CD26high T cells elicit tumor immunity against multiple malignancies via enhanced migration and persistence
Article Snippet: Flow cytometry Antibodies for extracellular stains were incubated with cells for 20 min in FACS buffer (PBS + 2%FBS). .. For intracellular staining, cells were activated with PMA/Ionomycin for 1 h, combined with Monensin (BioLegend) and incubated another 3 h prior to staining in Fix and Perm buffers (BioLegend). .. For transcription factors, the FOXP3 kit (BioLegend) was used according to manufacturer’s protocol.

Article Title: Partial Regulatory T Cell Depletion Prior to Acute Feline Immunodeficiency Virus Infection Does Not Alter Disease Pathogenesis
Article Snippet: Anti-mouse IgG3-APC (Jackson ImmunoResearch, West Grove, PA), anti-mouse IgG3-APC-Cy7 (Southern Biotech), and streptavidin-Pacific Orange (Invitrogen) were used for secondary detection. .. Intracellular FoxP3 staining was performed with eBioscience FoxP3 staining buffers and FoxP3-PE-Cy7 (FJK-16s; San Diego, CA) according to manufacturer's protocol, with the exception that cells remain in the permeabilization/wash buffer no longer than 30 min. For intracellular cytokine staining, cells were incubated with 1× monensin (BioLegend, San Diego, CA) for six hr, labeled for surface markers, fixed with 4% paraformaldehyde, permeabilized with BD Cytofix/Cytoperm kit Perm/Wash buffer, and stained with anti-cytokine mAbs. .. For intracellular Ki67 staining, cells were first labeled for surface markers, incubated with BD Cytofix/Cytoperm for 15 min, washed with BD Perm/Wash buffer, incubated with BD Cytoperm Plus for 10 min, washed, and incubated with BD Cytofix/Cytoperm for 5 min.

Staining:

Article Title: Inhibition of Ubc13-mediated Ubiquitination by GPS2 Regulates Multiple Stages of B Cell Development *
Article Snippet: B cells purified from spleen of Gps2−/− and wild-type littermates were cultured at 106 cells/ml in RPMI 10% FBS, 100× penicillin/streptomycin, and treated with 1 μg/ml LPS (TLR4 ligand), 1 μ m of ODN 2395 (TLR9 ligand), or 1 μg/ml gardiquimod (TLR7 ligand) (Invivogen) for 4 h. Treatment with 10 μg/ml of purified rat anti-mouse IgM (BD Pharmingen) was performed in the same culture condition for different time courses. .. For IL-10 stimulation and phenotyping, spleen cells were stimulated for 5 h in the presence of 10 μg/ml LPS, 50 ng/ml phorbol 12-myristate 13-acetate, and 500 ng/ml ionomycin in presence of 2 μ m monensin as described previously ( ) stained for surface markers, fixed with IC fixation buffer (Biolegend), permeabilized with IC permeabilization/wash buffer (Biolegend), and stained for IL-10. .. 293T cells were cultured in 4.5 g DMEM/liter glucose, 10% FBS, and 100× penicillin/streptomycin.

Article Title: STAT5 promotes accessibility and is required for BATF-mediated plasticity at the Il9 locus
Article Snippet: Information on cytokines and antibodies for cell culture are listed in Supplementary Table . .. Flow cytometry For cytokine staining, cells were stimulated with PMA (50 ng/ml, Sigma) and ionomycin (500 ng/ml, Sigma) for 3 h followed by monensin (2 μM, Biolegend) for a total of 5 h at 37 °C. .. After stimulation, cells were stained with a fixable viability dye (eBioscience) and antibodies for surface markers for 30 min at 4 °C, before fixation with 4% formaldehyde for 10 min dark at room temperature.

Labeling:

Cell Stimulation:

Article Title: Human intestinal tissue-resident memory T cells comprise transcriptionally and functionally distinct subsets
Article Snippet: In brief, purified intestine-derived mononuclear cells were plated at approximately 106 /well in a 96-well U-bottom plate. .. Cell stimulation cocktail containing phorbol 12-myristate 13-acetate (PMA) and ionomycin (BioLegend) was added in accordance with manufacturer’s instructions in the presence of brefeldin A and monensin (both BioLegend). ..

Recombinant:

Article Title: Palmitoylation regulates the intracellular trafficking and stability of c-Met
Article Snippet: .. Monensin and brefeldin A were obtained from Biolegend (San Diego, CA), Y27637 from Stem Cell Technology (Vancouver, BC), C75, 2-hydroxymyristic acid, and 17-Octadecynoic Acid from Caymen Chemicals (Ann Arbor, MI), cycloheximide, recombinant EGF, geranylgeranyltransferase inhibitor, farnesyltransferase inhibitor, vinblastine, latrunculin, dansylcadaverine, ethylisopropyl amiloride, methyl-β-cyclodextrin, nystatin, and 2-bromopalmitate were obtained from Sigma-Aldrich (St. Louis, MO). .. Click-iT® reaction buffers, azido-homoalanine, azide-palmitate, and methionine-free RPMI, were obtained from Invitrogen (Carlsbad, CA).