Structured Review

R&D Systems recombinant human il 4
STAT6 activation is ROP16-dependent and much faster than <t>IL-4-induced</t> activation. A , parasite infection was synchronized by potassium shift as described under “Experimental Procedures.” Infected HFF cells were fixed 1 min postinvasion with methanol and stained with DAPI and the indicated antibodies. B , HFF cells were stimulated with IL-4 (50 ng/ml), fixed at the indicated time points, and stained with DAPI and the indicated antibodies.
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Images

1) Product Images from "Toxoplasma Rhoptry Protein 16 (ROP16) Subverts Host Function by Direct Tyrosine Phosphorylation of STAT6 *"

Article Title: Toxoplasma Rhoptry Protein 16 (ROP16) Subverts Host Function by Direct Tyrosine Phosphorylation of STAT6 *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.112359

STAT6 activation is ROP16-dependent and much faster than IL-4-induced activation. A , parasite infection was synchronized by potassium shift as described under “Experimental Procedures.” Infected HFF cells were fixed 1 min postinvasion with methanol and stained with DAPI and the indicated antibodies. B , HFF cells were stimulated with IL-4 (50 ng/ml), fixed at the indicated time points, and stained with DAPI and the indicated antibodies.
Figure Legend Snippet: STAT6 activation is ROP16-dependent and much faster than IL-4-induced activation. A , parasite infection was synchronized by potassium shift as described under “Experimental Procedures.” Infected HFF cells were fixed 1 min postinvasion with methanol and stained with DAPI and the indicated antibodies. B , HFF cells were stimulated with IL-4 (50 ng/ml), fixed at the indicated time points, and stained with DAPI and the indicated antibodies.

Techniques Used: Activation Assay, Infection, Staining

2) Product Images from "Enhanced Type 2 Immune Reactions by Increased IL-22/IL-22Ra1 Signaling in Chronic Rhinosinusitis With Nasal Polyps"

Article Title: Enhanced Type 2 Immune Reactions by Increased IL-22/IL-22Ra1 Signaling in Chronic Rhinosinusitis With Nasal Polyps

Journal: Allergy, Asthma & Immunology Research

doi: 10.4168/aair.2020.12.6.980

IgE-mediated production and secretion of IL-22 from LAD2 cells. (A) IL-22 was measured by ELISA in the supernatants of LAD2-cell culture stimulated with IgE/SA, IL-4, IL-13, and SEB (n = 3 for each group). (B) IL-22 was measured by ELISA in the supernatants of LAD2-cell culture stimulated in variable concentrations (0.1, 1, or 5 μg/mL) of IgE with SA treatment (n = 3 for each group). (C) Representative double immunostaining (red: tryptase and green: IL-22). Box indicates representative findings (× 200). Scale bar = 50 μm. IL, interleukin; ELISA, enzyme-linked immunosorbent assay; IgE, immunoglobulin E; SEB, Staphylococcal enterotoxin B; SA, streptavidin; Un, unstimulated control.
Figure Legend Snippet: IgE-mediated production and secretion of IL-22 from LAD2 cells. (A) IL-22 was measured by ELISA in the supernatants of LAD2-cell culture stimulated with IgE/SA, IL-4, IL-13, and SEB (n = 3 for each group). (B) IL-22 was measured by ELISA in the supernatants of LAD2-cell culture stimulated in variable concentrations (0.1, 1, or 5 μg/mL) of IgE with SA treatment (n = 3 for each group). (C) Representative double immunostaining (red: tryptase and green: IL-22). Box indicates representative findings (× 200). Scale bar = 50 μm. IL, interleukin; ELISA, enzyme-linked immunosorbent assay; IgE, immunoglobulin E; SEB, Staphylococcal enterotoxin B; SA, streptavidin; Un, unstimulated control.

Techniques Used: Enzyme-linked Immunosorbent Assay, Cell Culture, Double Immunostaining

3) Product Images from "Prothymosin ? and a prothymosin ?-derived peptide enhance TH1-type immune responses against defined HER-2/neu epitopes"

Article Title: Prothymosin ? and a prothymosin ?-derived peptide enhance TH1-type immune responses against defined HER-2/neu epitopes

Journal: BMC Immunology

doi: 10.1186/1471-2172-14-43

T cells stimulated with proTα- or proTα(100–109)-matured HER-2/neu-peptide-pulsed autologous DCs produce T H 1-type cytokines. Monocytes from HLA-A2+/DR4+ donors were differentiated to iDCs, matured for 48 h with TNF-α, proTα or proTα(100–109), pulsed with HER-2(9 369 ) and HER-2(15 776 ) epitopes and used to stimulate autologous T cells. Recovered CD4+ T cells were analyzed for intracellular production of IFN-γ, IL-2, IL-4 and IL-10, in the absence (−) or presence (+) of the HER-2/neu peptides. First row, DCs matured with TNF-α; middle row, DCs matured with proTα; bottom row, DCs matured with proTα (100–109). Numbers indicate percentage of positive cells for each cytokine on gated CD4+ T cells. Shown data are from one representative donor of 5 tested.
Figure Legend Snippet: T cells stimulated with proTα- or proTα(100–109)-matured HER-2/neu-peptide-pulsed autologous DCs produce T H 1-type cytokines. Monocytes from HLA-A2+/DR4+ donors were differentiated to iDCs, matured for 48 h with TNF-α, proTα or proTα(100–109), pulsed with HER-2(9 369 ) and HER-2(15 776 ) epitopes and used to stimulate autologous T cells. Recovered CD4+ T cells were analyzed for intracellular production of IFN-γ, IL-2, IL-4 and IL-10, in the absence (−) or presence (+) of the HER-2/neu peptides. First row, DCs matured with TNF-α; middle row, DCs matured with proTα; bottom row, DCs matured with proTα (100–109). Numbers indicate percentage of positive cells for each cytokine on gated CD4+ T cells. Shown data are from one representative donor of 5 tested.

Techniques Used:

ProTα or proTα(100–109) induce DC maturation. Monocytes were differentiated to immature DCs (iDCs) upon 5-day incubation with GM-CSF and IL-4, followed by 48 h exposure to LPS, TNF-α, proTα or proTα(100–109). Expression of surface HLA-DR, CD11b, CD80, CD83, CD86, CD40 and CD14 on iDCs and mature DCs are shown as mean fluorescence intensity (MFI) ± SD from 5 donors. * p
Figure Legend Snippet: ProTα or proTα(100–109) induce DC maturation. Monocytes were differentiated to immature DCs (iDCs) upon 5-day incubation with GM-CSF and IL-4, followed by 48 h exposure to LPS, TNF-α, proTα or proTα(100–109). Expression of surface HLA-DR, CD11b, CD80, CD83, CD86, CD40 and CD14 on iDCs and mature DCs are shown as mean fluorescence intensity (MFI) ± SD from 5 donors. * p

Techniques Used: Incubation, Expressing, Fluorescence

4) Product Images from "Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends to modulate antibody class-switch DNA recombination"

Article Title: Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends to modulate antibody class-switch DNA recombination

Journal: Nature Communications

doi: 10.1038/ncomms14244

Polθ −/− B cells undergo normal CSR in vitro and in vivo . ( a ) Polθ +/+ and Polθ −/− B cells were stimulated with LPS plus IL-4 (for CSR to IgG1), LPS alone (IgG3), LPS plus IFN-γ (IgG2c) or LPS plus TGF-β, IL-4, IL-5 and anti-δ mAb/dex (IgA). After a 96 h of culture, the B cells were analysed for surface IgG1, IgG3, IgG2c or IgA by flow cytometry. ( b ) Proliferation of Polθ +/+ and Polθ −/− B cells labelled with CFSE and stimulated by LPS plus IL-4 for 96 h. CFSE intensity and surface IgG1 expression analysed by flow cytometry. Proportion of surface IgG1 + B cells at each cell division indicated. P -values determined using a paired Student's t -test. Data are from one representative of three independent experiments. ( c ) Polθ +/+ and Polθ −/− B cells cultured with LPS or LPS plus IL-4 for 60 h. Expression of Polθ , Aicda , germline Iγ1-Cγ1 and Iγ3-Cγ3, circle Iγ1-Cμ and Iγ3-Cμ, and post-recombination Iμ-Cγ1 and Iμ-Cγ3 transcripts analysed by qRT–PCR and normalized to Gapdh transcript, depicted as relative to the expression of each transcript in Polθ +/+ B cells, set as 1. Each sample was run in triplicate. Data are from three independent experiments involving three pairs of Polθ +/+ and Polθ −/− mice (mean±s.d.). P -values determined using a paired Student's t -test. ( d , e ) Polθ +/+ and Polθ −/− littermates were injected with NP 16 -CGG and killed 10 days later. ( d ) Titres of circulating IgM, IgG1 and IgA analysed by enzyme-linked immunosorbent assay (ELISA), expressed as μgeq ml −1 . P -values determined using a paired Student's t -test. Data are from three independent experiments (mean±s.d.). ( e ) Surface IgG1 expression in spleen B220 + PNA hi GC B cells analysed by flow cytometry. Data are from one representative of three independent experiments.
Figure Legend Snippet: Polθ −/− B cells undergo normal CSR in vitro and in vivo . ( a ) Polθ +/+ and Polθ −/− B cells were stimulated with LPS plus IL-4 (for CSR to IgG1), LPS alone (IgG3), LPS plus IFN-γ (IgG2c) or LPS plus TGF-β, IL-4, IL-5 and anti-δ mAb/dex (IgA). After a 96 h of culture, the B cells were analysed for surface IgG1, IgG3, IgG2c or IgA by flow cytometry. ( b ) Proliferation of Polθ +/+ and Polθ −/− B cells labelled with CFSE and stimulated by LPS plus IL-4 for 96 h. CFSE intensity and surface IgG1 expression analysed by flow cytometry. Proportion of surface IgG1 + B cells at each cell division indicated. P -values determined using a paired Student's t -test. Data are from one representative of three independent experiments. ( c ) Polθ +/+ and Polθ −/− B cells cultured with LPS or LPS plus IL-4 for 60 h. Expression of Polθ , Aicda , germline Iγ1-Cγ1 and Iγ3-Cγ3, circle Iγ1-Cμ and Iγ3-Cμ, and post-recombination Iμ-Cγ1 and Iμ-Cγ3 transcripts analysed by qRT–PCR and normalized to Gapdh transcript, depicted as relative to the expression of each transcript in Polθ +/+ B cells, set as 1. Each sample was run in triplicate. Data are from three independent experiments involving three pairs of Polθ +/+ and Polθ −/− mice (mean±s.d.). P -values determined using a paired Student's t -test. ( d , e ) Polθ +/+ and Polθ −/− littermates were injected with NP 16 -CGG and killed 10 days later. ( d ) Titres of circulating IgM, IgG1 and IgA analysed by enzyme-linked immunosorbent assay (ELISA), expressed as μgeq ml −1 . P -values determined using a paired Student's t -test. Data are from three independent experiments (mean±s.d.). ( e ) Surface IgG1 expression in spleen B220 + PNA hi GC B cells analysed by flow cytometry. Data are from one representative of three independent experiments.

Techniques Used: In Vitro, In Vivo, Flow Cytometry, Cytometry, Expressing, Cell Culture, Quantitative RT-PCR, Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay

Knockdown of Ku86 expression in Rad52 −/− B cells virtually ablates CSR and enforced expression of Rad52 in normal B cells significantly impairs CSR. Rad52 −/− and Rad52 +/+ B cells were transduced with pGFP-C-Ku86-shRNALenti lentiviral vector expressing Ku86 -specific shRNA and GFP, or pGFP-C-scr-shRNALenti lentiviral vector expressing scrambled shRNA and GFP. Rad52 +/+ B cells were activated with LPS for 12 h and transduced with empty pMIG-GFP retroviral vector or pMIG-GFP-Rad52 retroviral vector expressing recombinant Rad52. After lentiviral or retroviral transduction, B cells were cultured for 96 h with LPS plus IL-4. ( a ) B-cell expression of Ku86, Rad52 and β-Actin proteins analysed by immunoblotting. ( b ) Proportions of surface IgG1 + B cells among pGFP-C-scr-shRNALenti or pGFP-C-Ku86-shRNALenti lentiviral vector-transduced (B220 + GFP + ) Rad52 −/− and Rad52 +/+ B cells were analysed by flow cytometry. Data are from one representative of three independent experiments. ( c ) Proportions of surface IgG1 + B cells among empty pMIG-GFP or pMIG-GFP-Rad52 retroviral vector-transduced (B220 + GFP + ) Rad52 +/+ B cells were analysed by flow cytometry. Data are from one representative of three independent experiments.
Figure Legend Snippet: Knockdown of Ku86 expression in Rad52 −/− B cells virtually ablates CSR and enforced expression of Rad52 in normal B cells significantly impairs CSR. Rad52 −/− and Rad52 +/+ B cells were transduced with pGFP-C-Ku86-shRNALenti lentiviral vector expressing Ku86 -specific shRNA and GFP, or pGFP-C-scr-shRNALenti lentiviral vector expressing scrambled shRNA and GFP. Rad52 +/+ B cells were activated with LPS for 12 h and transduced with empty pMIG-GFP retroviral vector or pMIG-GFP-Rad52 retroviral vector expressing recombinant Rad52. After lentiviral or retroviral transduction, B cells were cultured for 96 h with LPS plus IL-4. ( a ) B-cell expression of Ku86, Rad52 and β-Actin proteins analysed by immunoblotting. ( b ) Proportions of surface IgG1 + B cells among pGFP-C-scr-shRNALenti or pGFP-C-Ku86-shRNALenti lentiviral vector-transduced (B220 + GFP + ) Rad52 −/− and Rad52 +/+ B cells were analysed by flow cytometry. Data are from one representative of three independent experiments. ( c ) Proportions of surface IgG1 + B cells among empty pMIG-GFP or pMIG-GFP-Rad52 retroviral vector-transduced (B220 + GFP + ) Rad52 +/+ B cells were analysed by flow cytometry. Data are from one representative of three independent experiments.

Techniques Used: Expressing, Transduction, Plasmid Preparation, shRNA, Recombinant, Cell Culture, Flow Cytometry, Cytometry

Aicda ( AICDA )/CSR-inducing stimuli decrease expression of Rad52 ( RAD52 ) and increase expression of Ku70/Ku86 ( KU70/KU86 ) and Polθ ( POLθ ) in mouse and human B cells. ( a ) C57BL/6 B cells were stimulated with different amounts of LPS only, or LPS or mCD154 plus IL-4 for 0, 24, 48 and 72 h. Aicda , Rad52 , Polθ and Ku70 / Ku86 transcripts were analysed by real-time qRT–PCR. Expression of Aicda normalized to Cd79b expression, expression of Rad52 , Ku70/Ku86 and Polθ normalized to Gapdh expression and depicted relative to the expression in unstimulated B cells, set as 1. Expression of AID, Rad52, Ku70, Ku86, Polθ and β-Actin proteins in B cells stimulated with LPS (3.0 μg ml −1 ) plus IL-4 were analysed by immunoblotting. ( b ) Human naive B cells were stimulated with different amounts of mCD154 plus human IL-4 and human IL-21 for 0, 24, 48, 72 or 96 h. AICDA , RAD52 , KU70 / KU86 and POLθ transcripts were analysed by real-time qRT–PCR. Expression normalized to CD79b expression and depicted relative to the expression in unstimulated B cells, set as 1. P -values determined using a paired Student's t -test. Data are from one representative of three independent experiments.
Figure Legend Snippet: Aicda ( AICDA )/CSR-inducing stimuli decrease expression of Rad52 ( RAD52 ) and increase expression of Ku70/Ku86 ( KU70/KU86 ) and Polθ ( POLθ ) in mouse and human B cells. ( a ) C57BL/6 B cells were stimulated with different amounts of LPS only, or LPS or mCD154 plus IL-4 for 0, 24, 48 and 72 h. Aicda , Rad52 , Polθ and Ku70 / Ku86 transcripts were analysed by real-time qRT–PCR. Expression of Aicda normalized to Cd79b expression, expression of Rad52 , Ku70/Ku86 and Polθ normalized to Gapdh expression and depicted relative to the expression in unstimulated B cells, set as 1. Expression of AID, Rad52, Ku70, Ku86, Polθ and β-Actin proteins in B cells stimulated with LPS (3.0 μg ml −1 ) plus IL-4 were analysed by immunoblotting. ( b ) Human naive B cells were stimulated with different amounts of mCD154 plus human IL-4 and human IL-21 for 0, 24, 48, 72 or 96 h. AICDA , RAD52 , KU70 / KU86 and POLθ transcripts were analysed by real-time qRT–PCR. Expression normalized to CD79b expression and depicted relative to the expression in unstimulated B cells, set as 1. P -values determined using a paired Student's t -test. Data are from one representative of three independent experiments.

Techniques Used: Expressing, Quantitative RT-PCR

Rad52 deficiency reduces microhomologies at recombination S–S region junctions in vitro and in vivo . Histograms depict percentages of Sμ–Sγ1 junction sequences with indicated numbers of nucleotide overlaps in Rad52 +/+ ( n =30) and Rad52 −/− B cells ( n =30) stimulated with LPS plus IL-4 for 96 h (as in Supplementary Fig. 1 ), and Sμ–Sα junction sequences with indicated numbers of nucleotide overlaps (microhomologies) in Rad52 +/+ ( n =30) and Rad52 −/− B cells ( n =30) from the Peyer's patches of three pairs of Rad52 +/+ and Rad52 −/− C57BL/6 littermates (as in Supplementary Fig. 2 ). 0 indicates no microhomology. The average length of nucleotide overlap and the numbers of sequences analysed ( n ) are indicated. P -values determined using a paired Student's t -test.
Figure Legend Snippet: Rad52 deficiency reduces microhomologies at recombination S–S region junctions in vitro and in vivo . Histograms depict percentages of Sμ–Sγ1 junction sequences with indicated numbers of nucleotide overlaps in Rad52 +/+ ( n =30) and Rad52 −/− B cells ( n =30) stimulated with LPS plus IL-4 for 96 h (as in Supplementary Fig. 1 ), and Sμ–Sα junction sequences with indicated numbers of nucleotide overlaps (microhomologies) in Rad52 +/+ ( n =30) and Rad52 −/− B cells ( n =30) from the Peyer's patches of three pairs of Rad52 +/+ and Rad52 −/− C57BL/6 littermates (as in Supplementary Fig. 2 ). 0 indicates no microhomology. The average length of nucleotide overlap and the numbers of sequences analysed ( n ) are indicated. P -values determined using a paired Student's t -test.

Techniques Used: In Vitro, In Vivo

Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends. ( a ) Ku70/Ku86 and Rad52 are recruited to CSR-targeted S region DNA in an AID-dependent manner in B cells undergoing CSR. Aicda +/+ and Aicda −/− B cells were stimulated with LPS or LPS plus IL-4 for 60 h. Cross-linked chromatin was precipitated using rabbit anti-Rad52 antibody or mouse anti-Ku70/Ku86 mAb. Precipitated Sμ, Sγ1 and Sγ3 DNA quantified by real-time quantitative PCR (qPCR); amounts relative to those in Aicda +/+ B cells, set as 1. Each precipitated DNA sample was run as triplicate in qPCR; the average of each triplicate was used as data point for that individual sample. Data are from three independent experiments involving three pairs of Aicda +/+ and Aicda −/− mice (mean±s.d.). P -values determined using a paired Student's t -test. ( b ) Precipitated Sμ and Cμ DNA from Aicda +/+ and Aicda −/− B cells detected by PCR. Data are one representative of three independent experiments. ( c ) Rad52 +/+ and Rad52 −/− B cells were stimulated with LPS or LPS plus IL-4 for 60 h. Chromatin was cross-linked and precipitated using a mouse anti-Ku70/Ku86 mAb. The precipitated Sμ, Sγ1 and Sγ3 DNAs quantified by real-time qPCR; amounts in Rad52 −/− B cells are as relative to those in Rad52 +/+ B cells, set as 1. Each precipitated DNA sample was run as triplicate in qPCR; the average of each triplicate was used as data point for that individual sample. Data are from three independent experiments involving three pairs of Rad52 +/+ and Rad52 −/− mice (mean±s.d.). P -values determined using a paired Student's t -test. ( d ) Rad52 +/+ and Rad52 −/− B cells were stimulated with LPS or LPS plus IL-4 for 60 h. DNA ends were labeled in situ with bio-dUTP using TdT. Chromatin was cross-linked and precipitated using mouse anti-Ku70/Ku86 mAb (left panel) or rabbit anti-Rad52 antibody (right panel). After resuspension, DNA with broken ends was pulled down with streptavidin magnetic beads. Precipitated Sμ, Sγ1, Sγ3 and Cμ DNAs quantified by real-time qPCR (this approach allowed for detection of Ku70/Ku86 or Rad52 bound to DSB-free ends); amounts of precipitated DNA are relative to those in Rad52 +/+ B cells, set as 1 (left panel), or relative to respective input DNA (right panel). Each precipitated DNA sample was run as triplicate in qPCR; the average of each triplicate was used as data point for that individual sample. Data are from three independent experiments involving three pairs of Rad52 +/+ and Rad52 −/− mice (mean±s.d.). P -values determined using a paired Student's t -test. ( e , f ) EMSA using a biotin-labelled double-stranded Sμ DNA probe and ( e ) recombinant human RAD52 and KU70/KU86 (constant amount) proteins at different ratios (1:1, 2:1 and 4:1), or ( f ) nuclear extracts (10 μg protein) from Rad52 +/+ and Rad52 −/− B cells (stimulated with LPS plus IL-4 for 60 h) incubated with mouse anti-Ku70/Ku86 mAb or irrelevant mouse IgG (control). The formation of protein–DNA complexes was shifted by anti-Ku70/Ku86 mAb. Numbers below gel image indicate relative density of protein–DNA complex bands normalized with the density of free probe. Shown is one representative gel of three independent experiments.
Figure Legend Snippet: Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends. ( a ) Ku70/Ku86 and Rad52 are recruited to CSR-targeted S region DNA in an AID-dependent manner in B cells undergoing CSR. Aicda +/+ and Aicda −/− B cells were stimulated with LPS or LPS plus IL-4 for 60 h. Cross-linked chromatin was precipitated using rabbit anti-Rad52 antibody or mouse anti-Ku70/Ku86 mAb. Precipitated Sμ, Sγ1 and Sγ3 DNA quantified by real-time quantitative PCR (qPCR); amounts relative to those in Aicda +/+ B cells, set as 1. Each precipitated DNA sample was run as triplicate in qPCR; the average of each triplicate was used as data point for that individual sample. Data are from three independent experiments involving three pairs of Aicda +/+ and Aicda −/− mice (mean±s.d.). P -values determined using a paired Student's t -test. ( b ) Precipitated Sμ and Cμ DNA from Aicda +/+ and Aicda −/− B cells detected by PCR. Data are one representative of three independent experiments. ( c ) Rad52 +/+ and Rad52 −/− B cells were stimulated with LPS or LPS plus IL-4 for 60 h. Chromatin was cross-linked and precipitated using a mouse anti-Ku70/Ku86 mAb. The precipitated Sμ, Sγ1 and Sγ3 DNAs quantified by real-time qPCR; amounts in Rad52 −/− B cells are as relative to those in Rad52 +/+ B cells, set as 1. Each precipitated DNA sample was run as triplicate in qPCR; the average of each triplicate was used as data point for that individual sample. Data are from three independent experiments involving three pairs of Rad52 +/+ and Rad52 −/− mice (mean±s.d.). P -values determined using a paired Student's t -test. ( d ) Rad52 +/+ and Rad52 −/− B cells were stimulated with LPS or LPS plus IL-4 for 60 h. DNA ends were labeled in situ with bio-dUTP using TdT. Chromatin was cross-linked and precipitated using mouse anti-Ku70/Ku86 mAb (left panel) or rabbit anti-Rad52 antibody (right panel). After resuspension, DNA with broken ends was pulled down with streptavidin magnetic beads. Precipitated Sμ, Sγ1, Sγ3 and Cμ DNAs quantified by real-time qPCR (this approach allowed for detection of Ku70/Ku86 or Rad52 bound to DSB-free ends); amounts of precipitated DNA are relative to those in Rad52 +/+ B cells, set as 1 (left panel), or relative to respective input DNA (right panel). Each precipitated DNA sample was run as triplicate in qPCR; the average of each triplicate was used as data point for that individual sample. Data are from three independent experiments involving three pairs of Rad52 +/+ and Rad52 −/− mice (mean±s.d.). P -values determined using a paired Student's t -test. ( e , f ) EMSA using a biotin-labelled double-stranded Sμ DNA probe and ( e ) recombinant human RAD52 and KU70/KU86 (constant amount) proteins at different ratios (1:1, 2:1 and 4:1), or ( f ) nuclear extracts (10 μg protein) from Rad52 +/+ and Rad52 −/− B cells (stimulated with LPS plus IL-4 for 60 h) incubated with mouse anti-Ku70/Ku86 mAb or irrelevant mouse IgG (control). The formation of protein–DNA complexes was shifted by anti-Ku70/Ku86 mAb. Numbers below gel image indicate relative density of protein–DNA complex bands normalized with the density of free probe. Shown is one representative gel of three independent experiments.

Techniques Used: Binding Assay, Real-time Polymerase Chain Reaction, Mouse Assay, Polymerase Chain Reaction, Labeling, In Situ, Magnetic Beads, Recombinant, Incubation

Rad52 deficiency reduces c-Myc/IgH translocations and microhomologies at c-Myc–IgH junctions. B cells from p53 −/− Rad52 +/+ and p53 −/− Rad52 −/− mice were stimulated with LPS plus IL-4 for 96 h before genomic DNA isolation. ( a ) c-Myc/IgH translocations were identified by amplifying DNA using long-range nested PCR involving primers specific to the IgH and c-Myc locus, and verified by Southern blot hybridization with an IgH or c-Myc- specific probe. Each PCR assay was performed using template DNA from 10 6 cells. Twenty-five amplicons from p53 −/− Rad52 +/+ B cells and 25 amplicons from p53 −/− Rad52 −/− B cells are shown. The frequencies of c-Myc/IgH translocations per cell are indicated below the gel images. PCR amplification products that can be detected by both IgH and c-Myc DNA probes were from c-Myc/IgH translocations: 8 of 25 and 3 of 25 amplicons from p53 −/− Rad52 +/+ and p53 −/− Rad52 −/− B cells, respectively, contained PCR amplification products from c-Myc/IgH translocations. ( b ) Sequences of the c-Myc/IgH translocation junctions. Amplified c-Myc – IgH junctional DNAs were cloned and sequenced. Each sequence is compared with germline c-Myc (above) and IgH (below) sequences. Microhomologies are bold and underlined. Sequences containing microhomologies are in blue. Point mutations are in red. Data are from three pairs of mice.
Figure Legend Snippet: Rad52 deficiency reduces c-Myc/IgH translocations and microhomologies at c-Myc–IgH junctions. B cells from p53 −/− Rad52 +/+ and p53 −/− Rad52 −/− mice were stimulated with LPS plus IL-4 for 96 h before genomic DNA isolation. ( a ) c-Myc/IgH translocations were identified by amplifying DNA using long-range nested PCR involving primers specific to the IgH and c-Myc locus, and verified by Southern blot hybridization with an IgH or c-Myc- specific probe. Each PCR assay was performed using template DNA from 10 6 cells. Twenty-five amplicons from p53 −/− Rad52 +/+ B cells and 25 amplicons from p53 −/− Rad52 −/− B cells are shown. The frequencies of c-Myc/IgH translocations per cell are indicated below the gel images. PCR amplification products that can be detected by both IgH and c-Myc DNA probes were from c-Myc/IgH translocations: 8 of 25 and 3 of 25 amplicons from p53 −/− Rad52 +/+ and p53 −/− Rad52 −/− B cells, respectively, contained PCR amplification products from c-Myc/IgH translocations. ( b ) Sequences of the c-Myc/IgH translocation junctions. Amplified c-Myc – IgH junctional DNAs were cloned and sequenced. Each sequence is compared with germline c-Myc (above) and IgH (below) sequences. Microhomologies are bold and underlined. Sequences containing microhomologies are in blue. Point mutations are in red. Data are from three pairs of mice.

Techniques Used: Mouse Assay, DNA Extraction, Nested PCR, Southern Blot, Hybridization, Polymerase Chain Reaction, Amplification, Translocation Assay, Clone Assay, Sequencing

Rad52 deficiency increases CSR. ( a ) B cells purified from Rad52 +/+ and Rad52 −/− C57BL/6 littermates were stimulated with mCD154 or LPS plus IL-4 (for CSR to IgG1), LPS alone (IgG3), LPS plus IFN-γ (IgG2c) or LPS plus TGF-β, IL-4, IL-5 and anti-δ mAb/dex (IgA). Purified Aicda +/+ and Aicda −/− B cells were stimulated with LPS plus IL-4. After 96 h of culture, the cells were analysed for surface IgG1, IgG3, IgG2c or IgA by flow cytometry. ( b ) Proliferation of Rad52 +/+ and Rad52 −/− B cells labelled with CFSE and stimulated for 72 h with different amounts of LPS or mCD154 plus IL-4. Progressive left shift of fluorescence intensity indicates B220 + B cell division. ( c ) Proliferation of Rad52 +/+ and Rad52 −/− B cells labelled with the cell division tracking fluorochrome CFSE and stimulated by mCD154 plus IL-4 or LPS plus IL-4 for 96 h. CFSE intensity and surface IgG1 expression analysed by flow cytometry. Proportion of surface IgG1 + B cells at each cell division indicated. P -values determined using a paired Student's t -test. Data are from one representative (left panels of each condition) or mean±s.d. of three independent experiments (right panels of each condition). ( d ) Recombinant Sμ–Sγ1 or Sμ–Sγ3 DNAs analysed by digestion–circularization PCR (DC-PCR) using serially twofold diluted HindIII digested and T4 DNA ligase-ligated genomic DNA from Rad52 +/+ or Rad52 −/− B cells after stimulation with LPS or LPS plus IL-4 for 96 h. Gapdh was used as a control for ligation and DNA loading. Data are from one representative of three independent experiments. ( e ) Rad52 +/+ and Rad52 −/− B cells were cultured with LPS or LPS plus IL-4 for 60 h. Aicda , Rad52 , Ku70 , Ku86 , Polθ , germline Iγ1-Cγ1 and Iγ3-Cγ3, circle Iγ1-Cμ and Iγ3-Cμ, and post-recombination Iμ-Cγ1 and Iμ-Cγ3 transcripts analysed by real-time qRT–PCR. Each sample was run in triplicate; expression normalized to Cd79b expression and depicted as relative to the expression in Rad52 +/+ B cells, set as 1. Data are from three independent experiments involving three pairs of Rad52 +/+ and Rad52 −/− mice (mean±s.d.). P values determined using a paired Student's t -test. ( f ) Expression of AID, Rad52, Ku70, Ku86, Polθ and β-Actin proteins in unstimulated Rad52 +/+ and Rad52 −/− B cells or Rad52 +/+ and Rad52 −/− B cells stimulated with LPS plus IL-4 for 72 h were analysed by immunoblotting. Data are from one representative of three independent experiments.
Figure Legend Snippet: Rad52 deficiency increases CSR. ( a ) B cells purified from Rad52 +/+ and Rad52 −/− C57BL/6 littermates were stimulated with mCD154 or LPS plus IL-4 (for CSR to IgG1), LPS alone (IgG3), LPS plus IFN-γ (IgG2c) or LPS plus TGF-β, IL-4, IL-5 and anti-δ mAb/dex (IgA). Purified Aicda +/+ and Aicda −/− B cells were stimulated with LPS plus IL-4. After 96 h of culture, the cells were analysed for surface IgG1, IgG3, IgG2c or IgA by flow cytometry. ( b ) Proliferation of Rad52 +/+ and Rad52 −/− B cells labelled with CFSE and stimulated for 72 h with different amounts of LPS or mCD154 plus IL-4. Progressive left shift of fluorescence intensity indicates B220 + B cell division. ( c ) Proliferation of Rad52 +/+ and Rad52 −/− B cells labelled with the cell division tracking fluorochrome CFSE and stimulated by mCD154 plus IL-4 or LPS plus IL-4 for 96 h. CFSE intensity and surface IgG1 expression analysed by flow cytometry. Proportion of surface IgG1 + B cells at each cell division indicated. P -values determined using a paired Student's t -test. Data are from one representative (left panels of each condition) or mean±s.d. of three independent experiments (right panels of each condition). ( d ) Recombinant Sμ–Sγ1 or Sμ–Sγ3 DNAs analysed by digestion–circularization PCR (DC-PCR) using serially twofold diluted HindIII digested and T4 DNA ligase-ligated genomic DNA from Rad52 +/+ or Rad52 −/− B cells after stimulation with LPS or LPS plus IL-4 for 96 h. Gapdh was used as a control for ligation and DNA loading. Data are from one representative of three independent experiments. ( e ) Rad52 +/+ and Rad52 −/− B cells were cultured with LPS or LPS plus IL-4 for 60 h. Aicda , Rad52 , Ku70 , Ku86 , Polθ , germline Iγ1-Cγ1 and Iγ3-Cγ3, circle Iγ1-Cμ and Iγ3-Cμ, and post-recombination Iμ-Cγ1 and Iμ-Cγ3 transcripts analysed by real-time qRT–PCR. Each sample was run in triplicate; expression normalized to Cd79b expression and depicted as relative to the expression in Rad52 +/+ B cells, set as 1. Data are from three independent experiments involving three pairs of Rad52 +/+ and Rad52 −/− mice (mean±s.d.). P values determined using a paired Student's t -test. ( f ) Expression of AID, Rad52, Ku70, Ku86, Polθ and β-Actin proteins in unstimulated Rad52 +/+ and Rad52 −/− B cells or Rad52 +/+ and Rad52 −/− B cells stimulated with LPS plus IL-4 for 72 h were analysed by immunoblotting. Data are from one representative of three independent experiments.

Techniques Used: Purification, Flow Cytometry, Cytometry, Fluorescence, Expressing, Recombinant, Polymerase Chain Reaction, Ligation, Cell Culture, Quantitative RT-PCR, Mouse Assay

Rad52-mediated S-region DSB repair favours intra-S region DNA recombination. ( a ) Each S region consists of highly repetitive motifs, which can facilitate the formation of microhomologies, in particular within the S region core. As the characteristically repetitive sequences are virtually unique to S regions, DSB ends in the same S region are better suited substrates for Rad52-mediated complementary DNA single-strand annealing than those in two different S regions, such as Sμ and Sγ1. Repetitive sequence elements in human and mouse Sμ and Sγ1 that can potentially form microhomologies were identified by Pustell Matrix dot plot using MacVector software and are depicted by small dots. Thick lines indicate the core regions of Sμ and Sγ1. ( b ) Schematic representation of the detection of intra-S region recombination (deletion) in Sμ region by PCR amplification. DNA-amplified sequences of Sμ region that underwent intra-S region DNA recombination are shorter than those of Sμ in the germline configuration. ( c ) Rad52 +/+ and Rad52 −/− B cells were stimulated with LPS plus IL-4 for 96 h. Sμ region DNA was amplified by nested PCR. PCR amplification products were then cloned into the TOPO cloning vector. Sμ region sequences from individual clones amplified by PCR and resolved through a 0.8% agarose gel. PCR amplification products smaller than that amplified from the germline Sμ region DNAs (indicated by arrows) are from Sμ region DNAs that underwent intra-S region recombination, thereby deleting variable lengths of DNA: 14 of 30 Sμ region DNAs in Rad52 +/+ B cells and 4 out of 30 Sμ region DNAs in Rad52 −/− B cells underwent intra-S region recombination. Data are from three pairs of Rad52 +/+ and Rad52 −/− C57BL/6 mice.
Figure Legend Snippet: Rad52-mediated S-region DSB repair favours intra-S region DNA recombination. ( a ) Each S region consists of highly repetitive motifs, which can facilitate the formation of microhomologies, in particular within the S region core. As the characteristically repetitive sequences are virtually unique to S regions, DSB ends in the same S region are better suited substrates for Rad52-mediated complementary DNA single-strand annealing than those in two different S regions, such as Sμ and Sγ1. Repetitive sequence elements in human and mouse Sμ and Sγ1 that can potentially form microhomologies were identified by Pustell Matrix dot plot using MacVector software and are depicted by small dots. Thick lines indicate the core regions of Sμ and Sγ1. ( b ) Schematic representation of the detection of intra-S region recombination (deletion) in Sμ region by PCR amplification. DNA-amplified sequences of Sμ region that underwent intra-S region DNA recombination are shorter than those of Sμ in the germline configuration. ( c ) Rad52 +/+ and Rad52 −/− B cells were stimulated with LPS plus IL-4 for 96 h. Sμ region DNA was amplified by nested PCR. PCR amplification products were then cloned into the TOPO cloning vector. Sμ region sequences from individual clones amplified by PCR and resolved through a 0.8% agarose gel. PCR amplification products smaller than that amplified from the germline Sμ region DNAs (indicated by arrows) are from Sμ region DNAs that underwent intra-S region recombination, thereby deleting variable lengths of DNA: 14 of 30 Sμ region DNAs in Rad52 +/+ B cells and 4 out of 30 Sμ region DNAs in Rad52 −/− B cells underwent intra-S region recombination. Data are from three pairs of Rad52 +/+ and Rad52 −/− C57BL/6 mice.

Techniques Used: Sequencing, Software, Polymerase Chain Reaction, Amplification, Nested PCR, Clone Assay, Plasmid Preparation, Agarose Gel Electrophoresis, Mouse Assay

5) Product Images from "HER2-specific T lymphocytes kill both trastuzumab-resistant and trastuzumab-sensitive breast cell lines in vitro"

Article Title: HER2-specific T lymphocytes kill both trastuzumab-resistant and trastuzumab-sensitive breast cell lines in vitro

Journal: Chinese Journal of Cancer Research

doi: 10.1007/s11670-012-0143-6

IFN-γ and IL-4 levels in the supernatant of T lymphocytes co-cultured with breast cancer cells. The IFN-γ level of the group of HER2-specific T cells (AAV-T) against MDA-MB-453, was significantly higher than the spontaneous release of
Figure Legend Snippet: IFN-γ and IL-4 levels in the supernatant of T lymphocytes co-cultured with breast cancer cells. The IFN-γ level of the group of HER2-specific T cells (AAV-T) against MDA-MB-453, was significantly higher than the spontaneous release of

Techniques Used: Cell Culture, Multiple Displacement Amplification

6) Product Images from "An atopy-associated polymorphism in the ectodomain of the IL-4R? chain (V50) regulates the persistence of STAT6 phosphorylation 1"

Article Title: An atopy-associated polymorphism in the ectodomain of the IL-4R? chain (V50) regulates the persistence of STAT6 phosphorylation 1

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.0803266

M1 blocking of murine IL-4 stimulated V50-IL-4Rα U937 clones
Figure Legend Snippet: M1 blocking of murine IL-4 stimulated V50-IL-4Rα U937 clones

Techniques Used: Blocking Assay, Clone Assay

Cytokine washout analysis of STAT6 phosphorylation in IL-4 stimulated and AG490 treated I50- and V50-IL-4Rα U937 clones
Figure Legend Snippet: Cytokine washout analysis of STAT6 phosphorylation in IL-4 stimulated and AG490 treated I50- and V50-IL-4Rα U937 clones

Techniques Used: Clone Assay

Cytokine washout analysis of STAT6 phosphorylation in V50-IL-4RαU937 clones stimulated with IL-4 in the absence or presence of AG490
Figure Legend Snippet: Cytokine washout analysis of STAT6 phosphorylation in V50-IL-4RαU937 clones stimulated with IL-4 in the absence or presence of AG490

Techniques Used: Clone Assay

Prolonged binding of IL-4 to V50-IL-4Rα
Figure Legend Snippet: Prolonged binding of IL-4 to V50-IL-4Rα

Techniques Used: Binding Assay

Analysis of STAT6 phosphorylation and dephosphorylation in the presence or absence of IL-4
Figure Legend Snippet: Analysis of STAT6 phosphorylation and dephosphorylation in the presence or absence of IL-4

Techniques Used: De-Phosphorylation Assay

Cytokine washout analysis of STAT6 phosphorylation in IL-4 stimulated I50- and V50-IL-4Rα U937 clones
Figure Legend Snippet: Cytokine washout analysis of STAT6 phosphorylation in IL-4 stimulated I50- and V50-IL-4Rα U937 clones

Techniques Used: Clone Assay

U937 cells expressing the V50-IL-4Rα show prolonged elevation of CIS mRNA after a short IL-4 treatment followed by cytokine washout
Figure Legend Snippet: U937 cells expressing the V50-IL-4Rα show prolonged elevation of CIS mRNA after a short IL-4 treatment followed by cytokine washout

Techniques Used: Expressing

Cytokine washout analysis of STAT6 phosphorylation levels in IL-4 stimulated and M1 treated I50- and V50-IL-4Rα U937 clones
Figure Legend Snippet: Cytokine washout analysis of STAT6 phosphorylation levels in IL-4 stimulated and M1 treated I50- and V50-IL-4Rα U937 clones

Techniques Used: Clone Assay

7) Product Images from "NK-4 exerts selective regulatory effects on the activation and function of allergy-related Th2 cells"

Article Title: NK-4 exerts selective regulatory effects on the activation and function of allergy-related Th2 cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0199666

NK-4 down-regulates secretion of TARC by NHDF stimulated with both IL-4 and TNF-α. NHDF were stimulated with 10 ng/ml IL-4 and 5 ng/ml TNF-α in the presence or absence (control) of varying concentrations of NK-4, dexamethasone or FK-506 (A) for 48 h at 37°C. In experiments comparing NK-4 with suplatast tosilate (B), NHDF were exposed to test articles for 15 min before stimulation with IL-4 and TNF-α. TARC levels in the cultures of NHDF without stimulation were below detectable limits. Results are the means ± S.D. of triplicate cultures. Results are representative of three independent experiments with similar results. ** p
Figure Legend Snippet: NK-4 down-regulates secretion of TARC by NHDF stimulated with both IL-4 and TNF-α. NHDF were stimulated with 10 ng/ml IL-4 and 5 ng/ml TNF-α in the presence or absence (control) of varying concentrations of NK-4, dexamethasone or FK-506 (A) for 48 h at 37°C. In experiments comparing NK-4 with suplatast tosilate (B), NHDF were exposed to test articles for 15 min before stimulation with IL-4 and TNF-α. TARC levels in the cultures of NHDF without stimulation were below detectable limits. Results are the means ± S.D. of triplicate cultures. Results are representative of three independent experiments with similar results. ** p

Techniques Used:

NK-4 selectively down-regulates IL-4 production by primary splenic T cells in response to anti-CD3ε mAb. BALB/c moue spleen cells (1.2 x 10 6 cells/well) were stimulated with 5 μg/ml anti-CD3ε mAb in the presence or absence (control) of varying concentrations of NK-4 for 48 h at 37°C in 96-well plates. Concentrations of IFN-γ (A) and IL-4 (B) in culture supernatants were measured by ELISA. The ratios of IFN-γ to IL-4 are shown (C). Results are the means ± S.D. of triplicate cultures. Results are representative of two independent experiments with similar results. ** p
Figure Legend Snippet: NK-4 selectively down-regulates IL-4 production by primary splenic T cells in response to anti-CD3ε mAb. BALB/c moue spleen cells (1.2 x 10 6 cells/well) were stimulated with 5 μg/ml anti-CD3ε mAb in the presence or absence (control) of varying concentrations of NK-4 for 48 h at 37°C in 96-well plates. Concentrations of IFN-γ (A) and IL-4 (B) in culture supernatants were measured by ELISA. The ratios of IFN-γ to IL-4 are shown (C). Results are the means ± S.D. of triplicate cultures. Results are representative of two independent experiments with similar results. ** p

Techniques Used: Enzyme-linked Immunosorbent Assay

NK-4 inhibits the IL-4-driven alteration of cytokine expression profile in THP-1 cells. THP-1 cells were cultured with 4 ng/ml and 10 ng/ml IL-4 for 3 days at 37°C in RPMI1640 medium containing 1% FCS in 12-well culture plate (A to C). In a separate experiment, THP-1 cells were cultured with 10 ng/ml IL-4 in the presence or absence of varying concentrations of NK-4 (D to F). After pre-treatment with IL-4, THP-1 cells (1.2 x 10 5 cells/well) in fresh RPMI1640 medium containing 10% FCS were stimulated overnight with LPS and the levels of TNF-α and IL-10 in the culture supernatants were determined by specific ELISA. The ratios of TNF-α to IL-10 are shown (C and F). Results are the means ± S.D. of triplicate cultures. Results are representative of three independent experiments with similar results. * p
Figure Legend Snippet: NK-4 inhibits the IL-4-driven alteration of cytokine expression profile in THP-1 cells. THP-1 cells were cultured with 4 ng/ml and 10 ng/ml IL-4 for 3 days at 37°C in RPMI1640 medium containing 1% FCS in 12-well culture plate (A to C). In a separate experiment, THP-1 cells were cultured with 10 ng/ml IL-4 in the presence or absence of varying concentrations of NK-4 (D to F). After pre-treatment with IL-4, THP-1 cells (1.2 x 10 5 cells/well) in fresh RPMI1640 medium containing 10% FCS were stimulated overnight with LPS and the levels of TNF-α and IL-10 in the culture supernatants were determined by specific ELISA. The ratios of TNF-α to IL-10 are shown (C and F). Results are the means ± S.D. of triplicate cultures. Results are representative of three independent experiments with similar results. * p

Techniques Used: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay

NK-4 down-regulates secretion of eotaxin by NHDF in response to IL-4 and/or TNF-α. NHDF were stimulated with varying concentrations of IL-4 (A) or TNF-α (B) in the presence or absence (control) of 10 μM NK-4 for 48 h at 37°C. The effect of NK-4 on the secretion of eotaxin from NHDF stimulated with 10 ng/ml IL-4 and 5 ng/ml TNF-α was assessed (C and D). Concentrations of eotaxin in culture supernatants were measured by ELISA (A to C). Cell numbers of NHDF were determined by cell counting kit-8 (D). Results are the means ± S.D. of triplicate cultures. Results are representative of three independent experiments with similar results. * p
Figure Legend Snippet: NK-4 down-regulates secretion of eotaxin by NHDF in response to IL-4 and/or TNF-α. NHDF were stimulated with varying concentrations of IL-4 (A) or TNF-α (B) in the presence or absence (control) of 10 μM NK-4 for 48 h at 37°C. The effect of NK-4 on the secretion of eotaxin from NHDF stimulated with 10 ng/ml IL-4 and 5 ng/ml TNF-α was assessed (C and D). Concentrations of eotaxin in culture supernatants were measured by ELISA (A to C). Cell numbers of NHDF were determined by cell counting kit-8 (D). Results are the means ± S.D. of triplicate cultures. Results are representative of three independent experiments with similar results. * p

Techniques Used: Enzyme-linked Immunosorbent Assay, Cell Counting

NK-4 selectively down-regulates Th2 cytokine production by antigen-stimulated established Th2 cells. Th1 clone #4 (2.5 x 10 4 cells/well) were stimulated with OVA (200 μg/ml) and MMC-treated BALB/c mouse spleen cells (1.2 x 10 6 cells/well) in the presence or absence (control) of varying concentrations of NK-4 for 48 h at 37°C in 96-well plates (A). D10.G4.1 cells (2.5 x 10 4 cells/well) were stimulated with MMC-treated C57BL/6 mouse spleen cells (1.2 x 10 6 cells/well) (B and C). Concentrations of IFN-γ (A), IL-4 (B) and IL-5 (C) in culture supernatants were measured by ELISA. Results are the means ± S.D. of triplicate cultures. Results are representative of three independent experiments with similar results. ** p
Figure Legend Snippet: NK-4 selectively down-regulates Th2 cytokine production by antigen-stimulated established Th2 cells. Th1 clone #4 (2.5 x 10 4 cells/well) were stimulated with OVA (200 μg/ml) and MMC-treated BALB/c mouse spleen cells (1.2 x 10 6 cells/well) in the presence or absence (control) of varying concentrations of NK-4 for 48 h at 37°C in 96-well plates (A). D10.G4.1 cells (2.5 x 10 4 cells/well) were stimulated with MMC-treated C57BL/6 mouse spleen cells (1.2 x 10 6 cells/well) (B and C). Concentrations of IFN-γ (A), IL-4 (B) and IL-5 (C) in culture supernatants were measured by ELISA. Results are the means ± S.D. of triplicate cultures. Results are representative of three independent experiments with similar results. ** p

Techniques Used: Enzyme-linked Immunosorbent Assay

NK-4 suppresses the STAT6 signaling pathway in NHDF stimulated with IL-4 and TNF-α. NHDF were grown in confluent monolayer cultures in 12-well plates and were stimulated with 10 ng/ml IL-4 and 5 ng/ml TNF-α in the presence or absence of varying concentrations of NK-4 for 15 min. Phosphorylation of STAT6 was determined by immunoblotting whole-cell lysates using specific antibodies against the phosphorylated or total STAT6 protein. A representative blot is shown (A). The optical density ratio of phospho-STAT6 to total STAT6 is shown (B). Data from three independent experiments were combined and expressed as the means ± SD. ** p
Figure Legend Snippet: NK-4 suppresses the STAT6 signaling pathway in NHDF stimulated with IL-4 and TNF-α. NHDF were grown in confluent monolayer cultures in 12-well plates and were stimulated with 10 ng/ml IL-4 and 5 ng/ml TNF-α in the presence or absence of varying concentrations of NK-4 for 15 min. Phosphorylation of STAT6 was determined by immunoblotting whole-cell lysates using specific antibodies against the phosphorylated or total STAT6 protein. A representative blot is shown (A). The optical density ratio of phospho-STAT6 to total STAT6 is shown (B). Data from three independent experiments were combined and expressed as the means ± SD. ** p

Techniques Used:

NK-4 selectively down-regulates Th2 cytokine production by anti-CD3ε mAb-stimulated established Th2 cells. Th1 clone #4 (A and B) and Th2 clone D10.G4.1 cells (C to E) (2.5 x 10 4 cells/well) were stimulated with immobilized anti-CD3ε mAb (8 μg/ml) in the presence or absence (control) of varying concentrations of NK-4 for 48 h at 37°C in 96-well plates. Concentrations of IFN-γ (A), IL-4 (C) and IL-5 (D) in culture supernatants were measured by ELISA. Cell numbers of #4 (B) and D10.G4.1 (E) were determined by cell counting kit-8. Results are the means ± S.D. of triplicate cultures. Results are representative of three independent experiments with similar results. * p
Figure Legend Snippet: NK-4 selectively down-regulates Th2 cytokine production by anti-CD3ε mAb-stimulated established Th2 cells. Th1 clone #4 (A and B) and Th2 clone D10.G4.1 cells (C to E) (2.5 x 10 4 cells/well) were stimulated with immobilized anti-CD3ε mAb (8 μg/ml) in the presence or absence (control) of varying concentrations of NK-4 for 48 h at 37°C in 96-well plates. Concentrations of IFN-γ (A), IL-4 (C) and IL-5 (D) in culture supernatants were measured by ELISA. Cell numbers of #4 (B) and D10.G4.1 (E) were determined by cell counting kit-8. Results are the means ± S.D. of triplicate cultures. Results are representative of three independent experiments with similar results. * p

Techniques Used: Enzyme-linked Immunosorbent Assay, Cell Counting

8) Product Images from "Effects of Human Respiratory Syncytial Virus, Metapneumovirus, Parainfluenza Virus 3 and Influenza Virus on CD4+ T Cell Activation by Dendritic Cells"

Article Title: Effects of Human Respiratory Syncytial Virus, Metapneumovirus, Parainfluenza Virus 3 and Influenza Virus on CD4+ T Cell Activation by Dendritic Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0015017

Cytokine expression by CD4+ T lymphocytes proliferating in response to MDDC treated with SEB or virus. This is a continuation of the experiment shown in Figure 2 . Expression of IFN-γ, IL-4, and/or TNF-α individually and in each double or triple combination (Boolean gating) was determined in live, proliferating, singlet CD4+ T cells. (A, B) Time course of production of IFN-γ and/or TNF-α by proliferating CD4+ T cells proliferating in response to MDDC that were mock-treated or treated with SEB, or live or UV-inactivated rHRSV or IAV, using cells from the same donor shown in Figure 2 A and B. (A) Total number of proliferating CD4+ T cells expressing IFN-γ or TNF-α ~ (B) Percentages of proliferating CD4+ T cells expressing IFN-γ and/or TNF-α, shown as pie charts. (C) Expression of IFN-γ and/or TNF-α by live, proliferated CD4+ T cells in experiments representing five of the eight donors shown in Figure 2 C. Values are expressed as percentages of total proliferated CD4+ T cells. The box plots show the median (horizontal line) flanked by the 2 nd and 3 rd quartile. The outer bars show the range of values. No statistical differences were observed between the treatments. (D) Comparison of the MFI of IFN-γ (left panel) or TNF-α (right panel) by proliferating CD4+ T cells that were positive for only the single cytokine versus those positive for both cytokines. The data are from the experiment in part C (cells from five donors). Statistical differences are indicated by asterisks (Friedman test with Dunns post hoc test (see Materials and Methods ); * = p≤0.05.
Figure Legend Snippet: Cytokine expression by CD4+ T lymphocytes proliferating in response to MDDC treated with SEB or virus. This is a continuation of the experiment shown in Figure 2 . Expression of IFN-γ, IL-4, and/or TNF-α individually and in each double or triple combination (Boolean gating) was determined in live, proliferating, singlet CD4+ T cells. (A, B) Time course of production of IFN-γ and/or TNF-α by proliferating CD4+ T cells proliferating in response to MDDC that were mock-treated or treated with SEB, or live or UV-inactivated rHRSV or IAV, using cells from the same donor shown in Figure 2 A and B. (A) Total number of proliferating CD4+ T cells expressing IFN-γ or TNF-α ~ (B) Percentages of proliferating CD4+ T cells expressing IFN-γ and/or TNF-α, shown as pie charts. (C) Expression of IFN-γ and/or TNF-α by live, proliferated CD4+ T cells in experiments representing five of the eight donors shown in Figure 2 C. Values are expressed as percentages of total proliferated CD4+ T cells. The box plots show the median (horizontal line) flanked by the 2 nd and 3 rd quartile. The outer bars show the range of values. No statistical differences were observed between the treatments. (D) Comparison of the MFI of IFN-γ (left panel) or TNF-α (right panel) by proliferating CD4+ T cells that were positive for only the single cytokine versus those positive for both cytokines. The data are from the experiment in part C (cells from five donors). Statistical differences are indicated by asterisks (Friedman test with Dunns post hoc test (see Materials and Methods ); * = p≤0.05.

Techniques Used: Expressing

Cytokine expression by proliferating CD4+ T lymphocytes co-cultured with virus-treated MDDC and SEB. (A) Time course of cytokine production by CD4+ T cells proliferating in response to MDDC that had been mock-treated or treated with live or UV-inactivated rHRSV, or live IAV, and co-cultured in the presence of SEB, using cells from the same donor represented in Figure 4 A. The percentages of live proliferated cells positive for IFN-γ+, TNF-α+, IFN-γ+ plus TNF-α+ and IFN-γ+ plus TNF-α + plus IL-4+, are shown in the pie charts. (B) Percentage of live, proliferating CD4+ T cells positive for IFN-γ (left panel), TNF-α (middle panel), and IFN-γ plus TNF-α (right panel) after four days of co-culture with SEB and MDDC treated with each of the four live or UV-inactivated viruses or mock treatment, using cells from the same six donors shown in Figure 4 C. As a control, one co-culture for each donor containing mock-treated MDDC also contained 75 IU/ml IFN-β in addition to SEB. The box plots show the median (horizontal line) flanked by the 2 nd and 3 rd quartile. The outer bars show the range of values. (C) Comparison of the MFI of IFN-γ (left panel) or TNF-α (right panel) by proliferating CD4+ T cells that were positive for only the single cytokine versus those positive for both cytokines. The data are from the experiment in part B (cells from six donors). Treatments sharing the same lowercase letter do not differ significantly.
Figure Legend Snippet: Cytokine expression by proliferating CD4+ T lymphocytes co-cultured with virus-treated MDDC and SEB. (A) Time course of cytokine production by CD4+ T cells proliferating in response to MDDC that had been mock-treated or treated with live or UV-inactivated rHRSV, or live IAV, and co-cultured in the presence of SEB, using cells from the same donor represented in Figure 4 A. The percentages of live proliferated cells positive for IFN-γ+, TNF-α+, IFN-γ+ plus TNF-α+ and IFN-γ+ plus TNF-α + plus IL-4+, are shown in the pie charts. (B) Percentage of live, proliferating CD4+ T cells positive for IFN-γ (left panel), TNF-α (middle panel), and IFN-γ plus TNF-α (right panel) after four days of co-culture with SEB and MDDC treated with each of the four live or UV-inactivated viruses or mock treatment, using cells from the same six donors shown in Figure 4 C. As a control, one co-culture for each donor containing mock-treated MDDC also contained 75 IU/ml IFN-β in addition to SEB. The box plots show the median (horizontal line) flanked by the 2 nd and 3 rd quartile. The outer bars show the range of values. (C) Comparison of the MFI of IFN-γ (left panel) or TNF-α (right panel) by proliferating CD4+ T cells that were positive for only the single cytokine versus those positive for both cytokines. The data are from the experiment in part B (cells from six donors). Treatments sharing the same lowercase letter do not differ significantly.

Techniques Used: Expressing, Cell Culture, Co-Culture Assay

CD4+ T lymphocyte gating strategy. DDAO-labeled immature MDDC were exposed to virus or controls for 4–6 h, co-cultured for up to 1 week with autologous purified CSFE-labeled CD4+ T cells, and stimulated with PMA and ionomycin prior to staining for flow cytometry. Dead cells were excluded using a live/dead stain and MDDC were excluded based on the DDAO tracer (a). T cells were identified by gating on CD3+ cells (b). Single cells were identified using forward scatter height (FSC-H) and forward scatter area (FSC-A) to analyze cell size (c). Remaining debris was removed using FSC-A to analyze size and side scatter (SSC-A) to analyze cell complexity (d). Proliferating T cells were quantified by gating on CFSE to monitor the dilution of this tracer that occurs with cell division (e; proliferated cells are boxed). Live, proliferating, singlet CD3+ cells were analyzed for the expression of IFN-γ (f), IL-4 (g), and TNF-α (h). Boolean gating was used to quantify subsets listed at the bottom.
Figure Legend Snippet: CD4+ T lymphocyte gating strategy. DDAO-labeled immature MDDC were exposed to virus or controls for 4–6 h, co-cultured for up to 1 week with autologous purified CSFE-labeled CD4+ T cells, and stimulated with PMA and ionomycin prior to staining for flow cytometry. Dead cells were excluded using a live/dead stain and MDDC were excluded based on the DDAO tracer (a). T cells were identified by gating on CD3+ cells (b). Single cells were identified using forward scatter height (FSC-H) and forward scatter area (FSC-A) to analyze cell size (c). Remaining debris was removed using FSC-A to analyze size and side scatter (SSC-A) to analyze cell complexity (d). Proliferating T cells were quantified by gating on CFSE to monitor the dilution of this tracer that occurs with cell division (e; proliferated cells are boxed). Live, proliferating, singlet CD3+ cells were analyzed for the expression of IFN-γ (f), IL-4 (g), and TNF-α (h). Boolean gating was used to quantify subsets listed at the bottom.

Techniques Used: Labeling, Cell Culture, Purification, Staining, Flow Cytometry, Cytometry, Expressing

9) Product Images from "IL-25 induces airways angiogenesis and expression of multiple angiogenic factors in a murine asthma model"

Article Title: IL-25 induces airways angiogenesis and expression of multiple angiogenic factors in a murine asthma model

Journal: Respiratory Research

doi: 10.1186/s12931-015-0197-3

IL-25, but not IL-4, IL-5 or IL-13 induced angiogenesis in vitro. Top panel: representative light photomicrographs (4x original magnification) show formation of primitive vascular tubule structures by human vascular endothelial cells after 11 days of culture (top panel) with medium (A) , VEGF (10 ng/mL) (B) , IL-25 (10 ng/mL) (C) , IL-4 (10 ng/mL) (D) , IL-5 (10 ng/mL) (E) and IL-13 (10 ng/mL) (F) . Bottom panel: computer-assisted quantification of total tubule lengths (G) , numbers of branch points (H) and total numbers of tubules (I) . Bars show the mean ± SEM of three separate experiments performed in duplicate. *p
Figure Legend Snippet: IL-25, but not IL-4, IL-5 or IL-13 induced angiogenesis in vitro. Top panel: representative light photomicrographs (4x original magnification) show formation of primitive vascular tubule structures by human vascular endothelial cells after 11 days of culture (top panel) with medium (A) , VEGF (10 ng/mL) (B) , IL-25 (10 ng/mL) (C) , IL-4 (10 ng/mL) (D) , IL-5 (10 ng/mL) (E) and IL-13 (10 ng/mL) (F) . Bottom panel: computer-assisted quantification of total tubule lengths (G) , numbers of branch points (H) and total numbers of tubules (I) . Bars show the mean ± SEM of three separate experiments performed in duplicate. *p

Techniques Used: In Vitro

10) Product Images from "Cytokines TNF-α, IL-6, IL-17F, and IL-4 Differentially Affect Osteogenic Differentiation of Human Adipose Stem Cells"

Article Title: Cytokines TNF-α, IL-6, IL-17F, and IL-4 Differentially Affect Osteogenic Differentiation of Human Adipose Stem Cells

Journal: Stem Cells International

doi: 10.1155/2016/1318256

Comparative analysis of the effect of pro- and anti-inflammatory cytokines on osteogenic differentiation of hASCs. ASCs were stimulated for 72 h with proinflammatory cytokines TNF- α , IL-6, IL-8, and IL-17F and the anti-inflammatory cytokine IL-4 (10 ng/mL). (a) TNF- α and IL-6 increased ALP activity at 7 days and IL-17F at 48 h and day 7. IL-4 increased ALP activity at day 4. Results are mean ± SD, n = 3. ∗ Significant effect of cytokine treatment, p
Figure Legend Snippet: Comparative analysis of the effect of pro- and anti-inflammatory cytokines on osteogenic differentiation of hASCs. ASCs were stimulated for 72 h with proinflammatory cytokines TNF- α , IL-6, IL-8, and IL-17F and the anti-inflammatory cytokine IL-4 (10 ng/mL). (a) TNF- α and IL-6 increased ALP activity at 7 days and IL-17F at 48 h and day 7. IL-4 increased ALP activity at day 4. Results are mean ± SD, n = 3. ∗ Significant effect of cytokine treatment, p

Techniques Used: ALP Assay, Activity Assay

Comparative analysis of the effect of pro- and anti-inflammatory cytokines on hASC proliferation. ASCs were stimulated for 72 h with proinflammatory cytokines TNF- α , IL-6, IL-8, and IL-17F and the anti-inflammatory cytokine IL-4 (10 ng/mL). (a) Gene expression of proliferation marker KI67 at 6 h, 48 h, and days 4 and 7. No significant effects of cytokines on KI67 expression were found, n = 7. (b) DNA content at 48 h, day 4, and day 7. TNF- α decreased DNA content at 48 h but increased DNA content at days 4 and 7. IL-17F decreased DNA content at day 4. n = 3, results are mean ± SD. ∗ Significant effect of cytokine treatment, p
Figure Legend Snippet: Comparative analysis of the effect of pro- and anti-inflammatory cytokines on hASC proliferation. ASCs were stimulated for 72 h with proinflammatory cytokines TNF- α , IL-6, IL-8, and IL-17F and the anti-inflammatory cytokine IL-4 (10 ng/mL). (a) Gene expression of proliferation marker KI67 at 6 h, 48 h, and days 4 and 7. No significant effects of cytokines on KI67 expression were found, n = 7. (b) DNA content at 48 h, day 4, and day 7. TNF- α decreased DNA content at 48 h but increased DNA content at days 4 and 7. IL-17F decreased DNA content at day 4. n = 3, results are mean ± SD. ∗ Significant effect of cytokine treatment, p

Techniques Used: Expressing, Marker

Comparative analysis of the effect of pro- and anti-inflammatory cytokines on osteogenic differentiation of hASCs. ASCs were stimulated for 72 h with proinflammatory cytokines TNF- α , IL-6, IL-8, and IL-17F and the anti-inflammatory cytokine IL-4 (10 ng/mL). (a) IL-4 decreased RUNX2 gene expression at days 4 and 7, n = 7. (b) TNF- α and IL-6 decreased gene expression of COL1 at day 4 and 48 h, n = 7. (c) Only IL-4, but not the other cytokines tested, increased osteocalcin gene expression at days 4 and 7. Results are mean ± SD, n = 7. ∗ Significant effect of cytokine treatment, p
Figure Legend Snippet: Comparative analysis of the effect of pro- and anti-inflammatory cytokines on osteogenic differentiation of hASCs. ASCs were stimulated for 72 h with proinflammatory cytokines TNF- α , IL-6, IL-8, and IL-17F and the anti-inflammatory cytokine IL-4 (10 ng/mL). (a) IL-4 decreased RUNX2 gene expression at days 4 and 7, n = 7. (b) TNF- α and IL-6 decreased gene expression of COL1 at day 4 and 48 h, n = 7. (c) Only IL-4, but not the other cytokines tested, increased osteocalcin gene expression at days 4 and 7. Results are mean ± SD, n = 7. ∗ Significant effect of cytokine treatment, p

Techniques Used: Expressing

11) Product Images from "Dendritic Cells Promoted by Ginseng Saponins Drive a Potent Th1 Polarization"

Article Title: Dendritic Cells Promoted by Ginseng Saponins Drive a Potent Th1 Polarization

Journal: Biomarker Insights

doi:

Effect of anti-IL-12 mAb on Th1 development induced by M1-, M4-, T-cadinol-, calamenene- or LPS-primed DC Allogeneic DC were co-cultured with naïve T cells at 1:5 DC/T cell ratio in the presence of control Ab or anti-IL-12 mAb (10 μg/ml). After 9 days of expansion in IL-2 expansion, T cells were counted and re-stimulated for 24 h on plates coated with anti-CD3/CD28. After 24 h, IFN-γ ( A ) and IL-4 ( B ) was measured by ELISA in culture supernatants. Data are the mean ± S.E.M. of five independent experiments.
Figure Legend Snippet: Effect of anti-IL-12 mAb on Th1 development induced by M1-, M4-, T-cadinol-, calamenene- or LPS-primed DC Allogeneic DC were co-cultured with naïve T cells at 1:5 DC/T cell ratio in the presence of control Ab or anti-IL-12 mAb (10 μg/ml). After 9 days of expansion in IL-2 expansion, T cells were counted and re-stimulated for 24 h on plates coated with anti-CD3/CD28. After 24 h, IFN-γ ( A ) and IL-4 ( B ) was measured by ELISA in culture supernatants. Data are the mean ± S.E.M. of five independent experiments.

Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay

Measurements of IFN-γ and IL-4 by ELISA in supernatant of naïve T cells stimulated by M1-, M4-, T-cadinol-, calamenene-, LPS-, CT-, or TNF-α-primed DC. Allogeneic DC were co-cultured for 5 days with naïve T cells at 1:5 DC/T cell ratio. After 9 days of expansion in IL-2, T cells were counted and re-stimulated for 24 h on plates coated with anti-CD3/CD28. IFN-γ ( A ) and IL-4 ( B ) were measured by ELISA in culture supernatants. Data are the mean ± S.E.M. of five independent experiments. *P
Figure Legend Snippet: Measurements of IFN-γ and IL-4 by ELISA in supernatant of naïve T cells stimulated by M1-, M4-, T-cadinol-, calamenene-, LPS-, CT-, or TNF-α-primed DC. Allogeneic DC were co-cultured for 5 days with naïve T cells at 1:5 DC/T cell ratio. After 9 days of expansion in IL-2, T cells were counted and re-stimulated for 24 h on plates coated with anti-CD3/CD28. IFN-γ ( A ) and IL-4 ( B ) were measured by ELISA in culture supernatants. Data are the mean ± S.E.M. of five independent experiments. *P

Techniques Used: Enzyme-linked Immunosorbent Assay, Cell Culture

M1-, M4- and TNF-α-primed DC induced the differentiation of naïve T cells to a Th1 response at 1:5 DC/T cell ratio After 9 days of expansion in IL-2 expansion, intracellular cytokine (IFN-γ and IL-4) concentrations were measured after re-stimulation with PMA and ionomycin for 5 h. Data are one experiment representative of four independent experiments.
Figure Legend Snippet: M1-, M4- and TNF-α-primed DC induced the differentiation of naïve T cells to a Th1 response at 1:5 DC/T cell ratio After 9 days of expansion in IL-2 expansion, intracellular cytokine (IFN-γ and IL-4) concentrations were measured after re-stimulation with PMA and ionomycin for 5 h. Data are one experiment representative of four independent experiments.

Techniques Used:

12) Product Images from "Ani9, A Novel Potent Small-Molecule ANO1 Inhibitor with Negligible Effect on ANO2"

Article Title: Ani9, A Novel Potent Small-Molecule ANO1 Inhibitor with Negligible Effect on ANO2

Journal: PLoS ONE

doi: 10.1371/journal.pone.0155771

Effect of Ani9 on endogenous CaCCs in PC3, Capan-1 and NHNE cells. A) Immunoblot of ANO1 protein in FRT, FRT-ANO1, PC3, Capan-1 and NHNE cells. Blots shown are representative of experiments performed three times. B-C) Effect of Ani9 on CaCCs activity was measured in PC3 and Capan-1 cells expressing a halide sensor YFP. The indicated concentrations of Ani9 were applied 20 min prior to CaCCs activation by 100 μM ATP. D) Summary of dose response (mean ± S.E., n = 6). E) Effect of Ani9 on CaCCs activity in IL-4 treated (10 ng/mL; 48h) NHNE cells. CaCCs currents were induced by 100 μM ATP and then the indicated concentrations of Ani9 were applied. The remaining CaCCs currents were abolished by 100 μM tannic acid (TA). (right) Summary of dose response (mean ± S.E., n = 4).
Figure Legend Snippet: Effect of Ani9 on endogenous CaCCs in PC3, Capan-1 and NHNE cells. A) Immunoblot of ANO1 protein in FRT, FRT-ANO1, PC3, Capan-1 and NHNE cells. Blots shown are representative of experiments performed three times. B-C) Effect of Ani9 on CaCCs activity was measured in PC3 and Capan-1 cells expressing a halide sensor YFP. The indicated concentrations of Ani9 were applied 20 min prior to CaCCs activation by 100 μM ATP. D) Summary of dose response (mean ± S.E., n = 6). E) Effect of Ani9 on CaCCs activity in IL-4 treated (10 ng/mL; 48h) NHNE cells. CaCCs currents were induced by 100 μM ATP and then the indicated concentrations of Ani9 were applied. The remaining CaCCs currents were abolished by 100 μM tannic acid (TA). (right) Summary of dose response (mean ± S.E., n = 4).

Techniques Used: Activity Assay, Expressing, Activation Assay

13) Product Images from "Increased nuclear suppressor of cytokine signaling 1 in asthmatic bronchial epithelium suppresses rhinovirus induction of innate interferons"

Article Title: Increased nuclear suppressor of cytokine signaling 1 in asthmatic bronchial epithelium suppresses rhinovirus induction of innate interferons

Journal: The Journal of Allergy and Clinical Immunology

doi: 10.1016/j.jaci.2014.11.039

Densitometry of SOCS1 Western blots in Fig 1 , effects of SOCS1 mRNA induction by rhinovirus after UV inactivation and filtration, and dose-dependent induction of SOCS1 mRNA by rhinovirus. A-C, Primary human BECs were treated with IL-4 or IL-13 (Fig E1, A ) or TNF-α or IL-1β (Fig E1, B ) or were infected with RV1B, RV16, or polyI:C (1 μg/mL; Fig E1, C ), and total protein was harvested over time and plotted versus α-tubulin as a control protein (n = 1 experiment for all induced SOCS1 protein). D and E, BECs were infected with RV16 (Fig E1, D ), UV-inactivated RV16, or filtered RV16, or RV1B (Fig E1, E ). F-RVIB , Filtered RV1B; UV-RV1B , UV-inactivated RV1B. UV-inactivated and rhinovirus-filtered (virus-free) preparations both showed a reduction in SOCS1 mRNA expression. F and G, BECs infected with RV16 (Fig E1, F ) or RV1B (Fig E1, G ) showed dose-dependent inductions of SOCS1 mRNA. * P
Figure Legend Snippet: Densitometry of SOCS1 Western blots in Fig 1 , effects of SOCS1 mRNA induction by rhinovirus after UV inactivation and filtration, and dose-dependent induction of SOCS1 mRNA by rhinovirus. A-C, Primary human BECs were treated with IL-4 or IL-13 (Fig E1, A ) or TNF-α or IL-1β (Fig E1, B ) or were infected with RV1B, RV16, or polyI:C (1 μg/mL; Fig E1, C ), and total protein was harvested over time and plotted versus α-tubulin as a control protein (n = 1 experiment for all induced SOCS1 protein). D and E, BECs were infected with RV16 (Fig E1, D ), UV-inactivated RV16, or filtered RV16, or RV1B (Fig E1, E ). F-RVIB , Filtered RV1B; UV-RV1B , UV-inactivated RV1B. UV-inactivated and rhinovirus-filtered (virus-free) preparations both showed a reduction in SOCS1 mRNA expression. F and G, BECs infected with RV16 (Fig E1, F ) or RV1B (Fig E1, G ) showed dose-dependent inductions of SOCS1 mRNA. * P

Techniques Used: Western Blot, Filtration, Infection, Expressing

SOCS1 mRNA and protein were induced in primary BECs by viruses and cytokines important in asthma pathogenesis. A, The T H 2 cytokines IL-4 and IL-13 both induced SOCS1 mRNA and protein in a time-dependent manner. B, The proinflammatory cytokines TNF-α and IL-1β also induced SOCS1 mRNA and protein in a time-dependent manner. C, RV1B, RV16, and 1 μg/mL polyI:C all induced SOCS1 mRNA and protein in a time-dependent manner. * P
Figure Legend Snippet: SOCS1 mRNA and protein were induced in primary BECs by viruses and cytokines important in asthma pathogenesis. A, The T H 2 cytokines IL-4 and IL-13 both induced SOCS1 mRNA and protein in a time-dependent manner. B, The proinflammatory cytokines TNF-α and IL-1β also induced SOCS1 mRNA and protein in a time-dependent manner. C, RV1B, RV16, and 1 μg/mL polyI:C all induced SOCS1 mRNA and protein in a time-dependent manner. * P

Techniques Used:

14) Product Images from "Modulation of Th1/Th2 Cytokine Balance by Quercetin In Vitro"

Article Title: Modulation of Th1/Th2 Cytokine Balance by Quercetin In Vitro

Journal: Medicines

doi: 10.3390/medicines7080046

Influence of quercetin on interferon (IFN)-γ production from human peripheral-blood CD4 + T cells in vitro. CD4 + T cells (1 × 10 6 cells/mL) were stimulated with 10.0 ng/mL IL-4 in the presence of various concentrations of quercetin for 24 h. IFN-γ levels in culture supernatants were examined by ELISA. The results were expressed as the mean pg/mL ± SE of five subjects.
Figure Legend Snippet: Influence of quercetin on interferon (IFN)-γ production from human peripheral-blood CD4 + T cells in vitro. CD4 + T cells (1 × 10 6 cells/mL) were stimulated with 10.0 ng/mL IL-4 in the presence of various concentrations of quercetin for 24 h. IFN-γ levels in culture supernatants were examined by ELISA. The results were expressed as the mean pg/mL ± SE of five subjects.

Techniques Used: In Vitro, Enzyme-linked Immunosorbent Assay

Influence of quercetin on mRNA expression for Th2-type cytokines in vitro. CD4 + T cells (1 × 10 6 cells/mL) were stimulated with 10.0 ng/mL IL-4 in the presence of various concentrations of quercetin for 4 h. mRNA expression for IL-5 ( a ) and IL-13 ( b ) was examined by real-time RT-PCR. The results were expressed as the mean cytokine/GAPDH ± SE of five subjects. * p > 0.05 versus IL-4 alone; ** p
Figure Legend Snippet: Influence of quercetin on mRNA expression for Th2-type cytokines in vitro. CD4 + T cells (1 × 10 6 cells/mL) were stimulated with 10.0 ng/mL IL-4 in the presence of various concentrations of quercetin for 4 h. mRNA expression for IL-5 ( a ) and IL-13 ( b ) was examined by real-time RT-PCR. The results were expressed as the mean cytokine/GAPDH ± SE of five subjects. * p > 0.05 versus IL-4 alone; ** p

Techniques Used: Expressing, In Vitro, Quantitative RT-PCR

Influence of quercetin on Th2-type cytokine production from human peripheral-blood CD4 + T cells in vitro. CD4 + T cells (1 × 10 6 cells/mL) were stimulated with 10.0 ng/mL IL-4 in the presence of various concentrations of quercetin for 24 h. Cytokine levels in culture supernatants were examined by ELISA. The results were expressed as the mean pg/mL ± SE of five subjects. ( a ): IL-5; ( b ): IL-13; * p > 0.05 versus IL-4 alone; ** p
Figure Legend Snippet: Influence of quercetin on Th2-type cytokine production from human peripheral-blood CD4 + T cells in vitro. CD4 + T cells (1 × 10 6 cells/mL) were stimulated with 10.0 ng/mL IL-4 in the presence of various concentrations of quercetin for 24 h. Cytokine levels in culture supernatants were examined by ELISA. The results were expressed as the mean pg/mL ± SE of five subjects. ( a ): IL-5; ( b ): IL-13; * p > 0.05 versus IL-4 alone; ** p

Techniques Used: In Vitro, Enzyme-linked Immunosorbent Assay

Influence of quercetin on transcription factor activation in CD4 + T cells in vitro. CD4 + T cells (1 × 10 6 cells/mL) were stimulated with 10.0 ng/mL IL-4 in the presence of various concentrations of quercetin for 1 h. Activation of transcription factors NF-κB ( a ) and STAT6 ( b ) was assessed by ELISA. The results were expressed as the mean OD at 450 nm ± SE of five subjects. * p > 0.05 versus IL-4 alone; ** p
Figure Legend Snippet: Influence of quercetin on transcription factor activation in CD4 + T cells in vitro. CD4 + T cells (1 × 10 6 cells/mL) were stimulated with 10.0 ng/mL IL-4 in the presence of various concentrations of quercetin for 1 h. Activation of transcription factors NF-κB ( a ) and STAT6 ( b ) was assessed by ELISA. The results were expressed as the mean OD at 450 nm ± SE of five subjects. * p > 0.05 versus IL-4 alone; ** p

Techniques Used: Activation Assay, In Vitro, Enzyme-linked Immunosorbent Assay

15) Product Images from "Human B cells accumulate immunoglobulin V gene somatic mutations in a cell contact-dependent manner in cultures supported by activated T cells but not in cultures supported by CD40 ligand"

Article Title: Human B cells accumulate immunoglobulin V gene somatic mutations in a cell contact-dependent manner in cultures supported by activated T cells but not in cultures supported by CD40 ligand

Journal: Clinical and Experimental Immunology

doi: 10.1046/j.1365-2249.1999.00919.x

Mutations accumulate in activated T cell-supported B cell cultures. Bars indicate percentage of rearrangements carrying mutations (± s.d. of three independent experiments). Libraries were made from pre-culture, purified B cells (top bar), or from 14 day B cell cultures which included either CD40L and IL-4 (second bar), resting T cells (third bar), or anti-CD3-activated T cells (bottom bar). V3–23 cDNA transcripts were identified by hybridization to probes M8 and/or M18. Mutation was scored as discordant hybridization to these probes. Each bar represents the analysis of at least 1000 V3–23 transcripts.
Figure Legend Snippet: Mutations accumulate in activated T cell-supported B cell cultures. Bars indicate percentage of rearrangements carrying mutations (± s.d. of three independent experiments). Libraries were made from pre-culture, purified B cells (top bar), or from 14 day B cell cultures which included either CD40L and IL-4 (second bar), resting T cells (third bar), or anti-CD3-activated T cells (bottom bar). V3–23 cDNA transcripts were identified by hybridization to probes M8 and/or M18. Mutation was scored as discordant hybridization to these probes. Each bar represents the analysis of at least 1000 V3–23 transcripts.

Techniques Used: Purification, Hybridization, Mutagenesis

16) Product Images from "Wear Particles Derived from Metal Hip Implants Induce the Generation of Multinucleated Giant Cells in a 3-Dimensional Peripheral Tissue-Equivalent Model"

Article Title: Wear Particles Derived from Metal Hip Implants Induce the Generation of Multinucleated Giant Cells in a 3-Dimensional Peripheral Tissue-Equivalent Model

Journal: PLoS ONE

doi: 10.1371/journal.pone.0124389

The level of IFN-γ was reduced while IL-10 level was increased in 10:1 treatment supernatants of day 14 culture. The 3-D model was prepared as previously stated, and supernatants were collected on days 4, 7, 9, and 14. Luminex Cytokine Th1/Th2 5-plex Immunoassay kit was used to measure the concentrations of IFN-γ, IL-2, IL-4, IL-5, and IL-10. The results are represented as mean +/- S.E. of three experimental samples from three independent experiments, n = 3. Asterisks, * = p
Figure Legend Snippet: The level of IFN-γ was reduced while IL-10 level was increased in 10:1 treatment supernatants of day 14 culture. The 3-D model was prepared as previously stated, and supernatants were collected on days 4, 7, 9, and 14. Luminex Cytokine Th1/Th2 5-plex Immunoassay kit was used to measure the concentrations of IFN-γ, IL-2, IL-4, IL-5, and IL-10. The results are represented as mean +/- S.E. of three experimental samples from three independent experiments, n = 3. Asterisks, * = p

Techniques Used: Luminex

Development of Peripheral Tissue-Equivalent Model using collagen gel / EA Hy926/ PBMCs and characterization of MGCs by flow cytometers. A) For the 3-D peripheral tissue equivalent model, particles were added at the time of collagen gel polymerization. Endothelial cells (EA, EAHy926 cell line) were grown on the top of the collagen gel to form a monolayer. One million peripheral blood mononuclear cells (PBMCs) were seeded on top of the endothelial cell monolayer and incubated for 2 weeks. MGCs or OCs were characterized and quantified on flow cytometer. Collagen gels were polymerized as described in method One million peripheral blood mononuclear cells (PBMCs) were treated without (B) or with (C) cytokines IL-4 and GMCSF for two weeks. Cells were harvested by digesting the gel with Collagenase. Cells were washed, fixed and permeabilized using BD cytofix/ perm buffer, and stained with propidium iodide before acquisition on a flow cytometer.
Figure Legend Snippet: Development of Peripheral Tissue-Equivalent Model using collagen gel / EA Hy926/ PBMCs and characterization of MGCs by flow cytometers. A) For the 3-D peripheral tissue equivalent model, particles were added at the time of collagen gel polymerization. Endothelial cells (EA, EAHy926 cell line) were grown on the top of the collagen gel to form a monolayer. One million peripheral blood mononuclear cells (PBMCs) were seeded on top of the endothelial cell monolayer and incubated for 2 weeks. MGCs or OCs were characterized and quantified on flow cytometer. Collagen gels were polymerized as described in method One million peripheral blood mononuclear cells (PBMCs) were treated without (B) or with (C) cytokines IL-4 and GMCSF for two weeks. Cells were harvested by digesting the gel with Collagenase. Cells were washed, fixed and permeabilized using BD cytofix/ perm buffer, and stained with propidium iodide before acquisition on a flow cytometer.

Techniques Used: Flow Cytometry, Incubation, Cytometry, Staining

17) Product Images from "c-Jun N-terminal kinase and Akt signalling pathways regulating tumour necrosis factor-α-induced interleukin-32 expression in human lung fibroblasts: implications in airway inflammation"

Article Title: c-Jun N-terminal kinase and Akt signalling pathways regulating tumour necrosis factor-α-induced interleukin-32 expression in human lung fibroblasts: implications in airway inflammation

Journal: Immunology

doi: 10.1111/imm.12374

Effects of a T helper type 1 (Th1) cytokine (interferon- γ ; IFN- γ ), Th2 cytokine (interleukin-4; IL-4), Th17 cytokine (IL-17A), or anti-inflammatory cytokine (IL-27) on tumour necrosis factor- α (TNF- α ) -induced IL-32 release
Figure Legend Snippet: Effects of a T helper type 1 (Th1) cytokine (interferon- γ ; IFN- γ ), Th2 cytokine (interleukin-4; IL-4), Th17 cytokine (IL-17A), or anti-inflammatory cytokine (IL-27) on tumour necrosis factor- α (TNF- α ) -induced IL-32 release

Techniques Used:

18) Product Images from "Interleukin-4 Distinctively Modifies Responses of Germinal Center-like and Activated B-cell-like Diffuse Large B-cell Lymphomas to Immuno-chemotherapy"

Article Title: Interleukin-4 Distinctively Modifies Responses of Germinal Center-like and Activated B-cell-like Diffuse Large B-cell Lymphomas to Immuno-chemotherapy

Journal: British journal of haematology

doi: 10.1111/j.1365-2141.2009.07851.x

IL-4 alters ability of DLBCL cell lines treated with chemotherapy to form colonies
Figure Legend Snippet: IL-4 alters ability of DLBCL cell lines treated with chemotherapy to form colonies

Techniques Used:

PI3K activation is necessary for the anti-apoptotic effects of IL-4 and stimulation of BCR in SUDHL-6 reverses effects of IL-4 on doxorubicin cytotoxicity
Figure Legend Snippet: PI3K activation is necessary for the anti-apoptotic effects of IL-4 and stimulation of BCR in SUDHL-6 reverses effects of IL-4 on doxorubicin cytotoxicity

Techniques Used: Activation Assay

IL-4 distinctively modified cytotoxic effects of doxorubicin in the ABC-like and GCB-like DLBCL cells
Figure Legend Snippet: IL-4 distinctively modified cytotoxic effects of doxorubicin in the ABC-like and GCB-like DLBCL cells

Techniques Used: Modification

IL-4 alters rituximab-mediated CDC but not ADCC of DLBCL cell lines
Figure Legend Snippet: IL-4 alters rituximab-mediated CDC but not ADCC of DLBCL cell lines

Techniques Used:

IL-4 alters expression of apoptosis-regulating proteins
Figure Legend Snippet: IL-4 alters expression of apoptosis-regulating proteins

Techniques Used: Expressing

19) Product Images from "Clostridium difficile Modulates Host Innate Immunity via Toxin-Independent and Dependent Mechanism(s)"

Article Title: Clostridium difficile Modulates Host Innate Immunity via Toxin-Independent and Dependent Mechanism(s)

Journal: PLoS ONE

doi: 10.1371/journal.pone.0069846

Generation of Th1/Th17 mediated immunity in response to C. difficile in an in-vitro murine model system. WT BMDCs were infected with PFA-fixed C. difficile strains. 24 h post-infection, stimulated BMDCs were co-cultured with WT splenocytes in the presence of anti-CD3/CD28. 96 h post co-culture, secreted cytokines IFN-γ (A), IL-17A (B), IL-10 (C), IL-4 (D) and IL-2 (E) were measured by ELISA. 1 µg/ml LPS stimulation served as reference control. Data represent mean ± SEM, n = 3. */ ∧ p
Figure Legend Snippet: Generation of Th1/Th17 mediated immunity in response to C. difficile in an in-vitro murine model system. WT BMDCs were infected with PFA-fixed C. difficile strains. 24 h post-infection, stimulated BMDCs were co-cultured with WT splenocytes in the presence of anti-CD3/CD28. 96 h post co-culture, secreted cytokines IFN-γ (A), IL-17A (B), IL-10 (C), IL-4 (D) and IL-2 (E) were measured by ELISA. 1 µg/ml LPS stimulation served as reference control. Data represent mean ± SEM, n = 3. */ ∧ p

Techniques Used: In Vitro, Infection, Cell Culture, Co-Culture Assay, Enzyme-linked Immunosorbent Assay

20) Product Images from "Cross-Talk between Human Mast Cells and Epithelial Cells by IgE-mediated Periostin Production in Eosinophilic Nasal Polyps"

Article Title: Cross-Talk between Human Mast Cells and Epithelial Cells by IgE-mediated Periostin Production in Eosinophilic Nasal Polyps

Journal: The Journal of allergy and clinical immunology

doi: 10.1016/j.jaci.2016.09.026

Cross-talk between human mast cells and epithelial cells in eosinophilic nasal polyps (A, B) IHC detection of periostin was performed within the NP tissues from patients with CRSwNP ( eosinophil, asterisk; mast cell, red arrow head; fibroblast, black arrow ). Black scale bar=10μm. (C) Representative double immunostaining (red: tryptase and blue: periostin). Arrows indicate double-positive immune cells (x1000). Black scale bar=10μm. (D) Counts of double-positive cells in NE-NP and E-NP (n=5 for each group). (E ) Periostin mRNA expression was measured by qRT-PCR in LAD2 cells stimulated with IgE, IL-4, IL-13 and TNF-α (n=9 for each group). (F) Periostin protein expression stimulated by IgE (n=3 for each group). (G) Number of integrin αV-positive epithelial cells per 100 cells was determined and averaged from three different areas of epithelium (n=10 for Control-UP, n=10 for CRSsNP-UP, n=10 for CRSwNP-UP, and n=37 for CRSwNP-NP) in IHC assays of tissue sections. The number of integrin αV-positive inflammatory cells was counted from five of the densest areas (HPF; x400) and averaged for each group. (H) Correlation between integrin αV and TSLP at the protein level. (I) Correlation between periostin and TSLP at the protein level. (J) Periostin-induced TSLP production in NHBE cells after Th2 cytokine and Poly(I:C) treatment (n=3 for each group). (K, L) Periostin-induced TSLP production was reversed by anti-integrin αVβ3 (K) and anti- integrin αVβ5 (L) (n=3 for each group). * P
Figure Legend Snippet: Cross-talk between human mast cells and epithelial cells in eosinophilic nasal polyps (A, B) IHC detection of periostin was performed within the NP tissues from patients with CRSwNP ( eosinophil, asterisk; mast cell, red arrow head; fibroblast, black arrow ). Black scale bar=10μm. (C) Representative double immunostaining (red: tryptase and blue: periostin). Arrows indicate double-positive immune cells (x1000). Black scale bar=10μm. (D) Counts of double-positive cells in NE-NP and E-NP (n=5 for each group). (E ) Periostin mRNA expression was measured by qRT-PCR in LAD2 cells stimulated with IgE, IL-4, IL-13 and TNF-α (n=9 for each group). (F) Periostin protein expression stimulated by IgE (n=3 for each group). (G) Number of integrin αV-positive epithelial cells per 100 cells was determined and averaged from three different areas of epithelium (n=10 for Control-UP, n=10 for CRSsNP-UP, n=10 for CRSwNP-UP, and n=37 for CRSwNP-NP) in IHC assays of tissue sections. The number of integrin αV-positive inflammatory cells was counted from five of the densest areas (HPF; x400) and averaged for each group. (H) Correlation between integrin αV and TSLP at the protein level. (I) Correlation between periostin and TSLP at the protein level. (J) Periostin-induced TSLP production in NHBE cells after Th2 cytokine and Poly(I:C) treatment (n=3 for each group). (K, L) Periostin-induced TSLP production was reversed by anti-integrin αVβ3 (K) and anti- integrin αVβ5 (L) (n=3 for each group). * P

Techniques Used: Immunohistochemistry, Double Immunostaining, Expressing, Quantitative RT-PCR

21) Product Images from "Follicular dendritic-like cells derived from human monocytes"

Article Title: Follicular dendritic-like cells derived from human monocytes

Journal: BMC Immunology

doi: 10.1186/1471-2172-6-23

Synergistic action of AP-inducing factors . Monocytes were cultured in the presence of a , 25 U/ml Il-4 and 3 U/ml GM-CSF; b , 0, 5 μM Dex; c , 3 U/ml GM-CSF; d , 25 U/ml Il-4, 3 U/ml GM-CSF and 0, 5 μM Dex (full mix). On day 15, the cultures were fixed and stained for expression of AP.
Figure Legend Snippet: Synergistic action of AP-inducing factors . Monocytes were cultured in the presence of a , 25 U/ml Il-4 and 3 U/ml GM-CSF; b , 0, 5 μM Dex; c , 3 U/ml GM-CSF; d , 25 U/ml Il-4, 3 U/ml GM-CSF and 0, 5 μM Dex (full mix). On day 15, the cultures were fixed and stained for expression of AP.

Techniques Used: Cell Culture, Staining, Expressing

AP expression in FDC-like cells . After 15 days of culture the presence of Il-4 (25 U/ml), GM-CSF (3 U/ml) and Dex (0, 5 μM), monocyte-derived cells were fixed and stained for AP (blue).
Figure Legend Snippet: AP expression in FDC-like cells . After 15 days of culture the presence of Il-4 (25 U/ml), GM-CSF (3 U/ml) and Dex (0, 5 μM), monocyte-derived cells were fixed and stained for AP (blue).

Techniques Used: Expressing, Derivative Assay, Staining

22) Product Images from "HGAL, a lymphoma prognostic biomarker, interacts with the cytoskeleton and mediates the effects of IL-6 on cell migration"

Article Title: HGAL, a lymphoma prognostic biomarker, interacts with the cytoskeleton and mediates the effects of IL-6 on cell migration

Journal: Blood

doi: 10.1182/blood-2007-04-087775

Pervanadate and IL-6 induce tyrosine phosphorylation of HGAL protein. SUDHL6 lymphoma cells were incubated for up to 60 minutes with pervanadate (1 mM) (A) or stimulated with IL-6 (10 ng/mL), IL-4 (10 ng/mL), anti-IgM (100 ng/mL), and IFNγ (1 μM) (B). Cellular lysates were extracted at the indicated time points, immunoprecipitated with anti-HGAL antibody, and blotted for phosphotyrosine (PTyr) and HGAL. Unconjugated beads served as a control (B). Representative blots of 3 independent experiments are shown.
Figure Legend Snippet: Pervanadate and IL-6 induce tyrosine phosphorylation of HGAL protein. SUDHL6 lymphoma cells were incubated for up to 60 minutes with pervanadate (1 mM) (A) or stimulated with IL-6 (10 ng/mL), IL-4 (10 ng/mL), anti-IgM (100 ng/mL), and IFNγ (1 μM) (B). Cellular lysates were extracted at the indicated time points, immunoprecipitated with anti-HGAL antibody, and blotted for phosphotyrosine (PTyr) and HGAL. Unconjugated beads served as a control (B). Representative blots of 3 independent experiments are shown.

Techniques Used: Incubation, Immunoprecipitation

23) Product Images from "B cell Sirt1 deacetylates histone and non-histone proteins for epigenetic modulation of AID expression and the antibody response"

Article Title: B cell Sirt1 deacetylates histone and non-histone proteins for epigenetic modulation of AID expression and the antibody response

Journal: Science Advances

doi: 10.1126/sciadv.aay2793

Aicda -inducing stimuli down-regulate Sirt1 in human and mouse B cells. ( A ) Sirt1-7 and Aicda expression in mouse naïve B cells before and after stimulation with LPS plus IL-4 for 72 hours, as measured by mRNA-Seq and depicted as RPKM (reads per kilobase of transcripts per million mapped reads; one of two independent experiments yielding comparable results). ( B ) Sirt1 and Aicda transcript levels [quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis] in mouse B cells stimulated with LPS or CD154 plus IL-4 for 0, 6, 12, 24, 48, or 72 hours. Data are ratios to the expression in unstimulated B cells (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates, left panel). Also shown are the inverse correlation scatter plots of Sirt1 and Aicda expression levels (right). ( C ) SIRT1 and AICDA expression (qRT-PCR analysis) in human naïve B cells stimulated with CD154 plus IL-4 and IL-21 for 0, 6, 12, 24, 48, 72, or 96 hours. Data are ratio to the expression in unstimulated B cells (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates, left). Also shown are the inverse correlation scatter plots of SIRT1 and AICDA expression levels (right). ( D ) Sirt1 and Aicda transcript levels (qRT-PCR analysis) in mouse B cells stimulated with anti-Igδ mAb or LPS plus IL-4 for 72 hours. Data are ratios to the expression in unstimulated B cells (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). ( E ) Sirt1 and Aicda expression (qRT-PCR analysis) in spleen B cells from C57BL/6 mice immunized with nil or NP 16 -CGG and analyzed 10 days after immunization. Data are ratios to the expression in nonimmunized mice (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). ( F ) Sirt1 and AID protein levels in CD19 + IgD hi GL7 − CD138 − naïve B cells, CD19 + IgD − GL7 hi CD138 − germinal center (GC) B cells, and CD19 lo CD138 hi plasma cells/plasmablasts in C57BL/6 mice immunized with NP 16 -CGG, as analyzed by flow cytometry 10 days after immunization. ( G to I ) Sirt1 and AID protein levels in mouse B cells before (nil) and after stimulation with LPS plus IL-4 for 72 hours analyzed by flow cytometry (G), intracellular immunofluorescence (H), and immunoblotting (I). Densitometry quantification of immunoblotting signals normalized to β-actin levels and depicted as ratios of readings in LPS plus IL-4 stimulated B cells to those in unstimulated (0 hours) B cells (I, right). Data in (F) to (I) are one of two independent experiments yielding similar results. *** P
Figure Legend Snippet: Aicda -inducing stimuli down-regulate Sirt1 in human and mouse B cells. ( A ) Sirt1-7 and Aicda expression in mouse naïve B cells before and after stimulation with LPS plus IL-4 for 72 hours, as measured by mRNA-Seq and depicted as RPKM (reads per kilobase of transcripts per million mapped reads; one of two independent experiments yielding comparable results). ( B ) Sirt1 and Aicda transcript levels [quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis] in mouse B cells stimulated with LPS or CD154 plus IL-4 for 0, 6, 12, 24, 48, or 72 hours. Data are ratios to the expression in unstimulated B cells (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates, left panel). Also shown are the inverse correlation scatter plots of Sirt1 and Aicda expression levels (right). ( C ) SIRT1 and AICDA expression (qRT-PCR analysis) in human naïve B cells stimulated with CD154 plus IL-4 and IL-21 for 0, 6, 12, 24, 48, 72, or 96 hours. Data are ratio to the expression in unstimulated B cells (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates, left). Also shown are the inverse correlation scatter plots of SIRT1 and AICDA expression levels (right). ( D ) Sirt1 and Aicda transcript levels (qRT-PCR analysis) in mouse B cells stimulated with anti-Igδ mAb or LPS plus IL-4 for 72 hours. Data are ratios to the expression in unstimulated B cells (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). ( E ) Sirt1 and Aicda expression (qRT-PCR analysis) in spleen B cells from C57BL/6 mice immunized with nil or NP 16 -CGG and analyzed 10 days after immunization. Data are ratios to the expression in nonimmunized mice (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). ( F ) Sirt1 and AID protein levels in CD19 + IgD hi GL7 − CD138 − naïve B cells, CD19 + IgD − GL7 hi CD138 − germinal center (GC) B cells, and CD19 lo CD138 hi plasma cells/plasmablasts in C57BL/6 mice immunized with NP 16 -CGG, as analyzed by flow cytometry 10 days after immunization. ( G to I ) Sirt1 and AID protein levels in mouse B cells before (nil) and after stimulation with LPS plus IL-4 for 72 hours analyzed by flow cytometry (G), intracellular immunofluorescence (H), and immunoblotting (I). Densitometry quantification of immunoblotting signals normalized to β-actin levels and depicted as ratios of readings in LPS plus IL-4 stimulated B cells to those in unstimulated (0 hours) B cells (I, right). Data in (F) to (I) are one of two independent experiments yielding similar results. *** P

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Mouse Assay, Flow Cytometry, Immunofluorescence

Sirt1 overexpression or activation dampens Aicda expression and CSR, while Sirt1 deletion further increases Aicda expression and CSR. ( A ) Ex vivo expression of Sirt1 and Aicda (qRT-PCR) in peripheral blood total B cells isolated from non-intentionally immunized Sirt1 +/+ and Sirt1 super mice. Data are ratios to expression in total B cells from Sirt1 +/+ mice (set as 1; means ± SEM of values from three Sirt1 +/+ mice and three Sirt1 super mice). ( B ) Serum titers [enzyme-linked immunosorbent assay (ELISA)] of total IgM, IgG1, and IgA in non-intentionally immunized Sirt1 +/+ and Sirt1 super mice (means ± SD of six Sirt1 +/+ mice and six Sirt1 super mice). ( C ) Expression of Sirt1, Aicda, Prdm1 , and Xbp1 transcripts (qRT-PCR) in Sirt1 +/+ (set as 1) and Sirt1 super B cells stimulated with LPS plus IL-4 for 72 hours (means ± SEM of three biological independent experiments, each consisting of triplicates; left) as well as immunoblotting and flow cytometry analysis of Sirt1 and AID protein levels in these cells (one of two independent experiments yielding similar results; right). ( D and E ) CSR (D) and plasma cell differentiation (E) in Sirt1 +/+ and Sirt1 super B cells (flow cytometry analysis) after stimulation with appropriate stimuli, as indicated, for 96 hours (left, one representative of three independent experiments; right, means ± SEM of three independent experiments). ( F ) CSR to IgG1 (flow cytometry analysis) in (B220 + GFP + ) B cells transduced by pMIG-GFP or pMIG-GFP-Sirt1 retrovirus (one of two independent experiments yielding similar results). ( G to I ) C57BL/6 B cells stimulated for 96 hours with LPS plus IL-4 in the presence of SRT1720 at indicated doses. Percentages of IgG1 + B cells and B220 low CD138 + plasma cells (G), cell proliferation [carboxyfluorescein diacetate succinimidyl ester (CFSE)–labeled cells] (H), and viability [7-aminoactinomycin D–negative (7-AAD − )] (I) (flow cytometry analysis). Data are means ± SEM of three independent experiments (G) or one representative of three independent experiments (H and I). ( J ) Aicda, Prdm1 , and Xbp1 transcript levels (qRT-PCR analysis) in purified B cells treated with nil or SRT1720 at the indicated doses and stimulated for 72 hours with LPS plus IL-4. Data are ratios to the expression in B cells treated with nil (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). ( K ) Sirt1 transcript levels (qRT-PCR analysis) in Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl B cells stimulated with LPS plus IL-4 for 0 and 72 hours. Data are ratios to the expression in unstimulated (0 hours) Aicda cre Sirt1 +/+ B cells (set as 1; ± SEM of three biological independent experiments, each consisting of triplicates). ( L ) CSR to different Ig isotypes (flow cytometry analysis) in Aidca cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl B cells stimulated for 96 hours with appropriate stimuli, as indicated. Data are one representative (left) and means ± SEM (right) of three independent experiments. ( M ) Expression of indicated genes (qRT-PCR analysis) in Aidca cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl B cells stimulated for 72 hours with LPS plus IL-4 or LPS plus TGF-β and anti-Igδ mAb-dextran (Iα-Cα and Iμ-Cα). Data are ratios to the expression in Aicda cre Sirt1 +/+ B cells (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). ( N ) AID protein levels in Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl B cells stimulated with LPS plus IL-4 for 72 hours as analyzed by intracellular staining and flow cytometry. Data are from one of two independent experiments yielding similar results. ( O ) Plasma cell differentiation (flow cytometry analysis) in Aidca cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl B cells stimulated for 96 hours with appropriate stimuli, as indicated. Data are one representative (left) and means ± SEM (right) of three biological independent experiments. ( P ) Proliferation of spleen B220 + B cells labeled with CFSE and stimulated with LPS plus IL-4 for 72 and 96 hours (top) and B cell viability (7-AAD − , bottom). Data are one representative of three independent experiments yielding similar results. * P
Figure Legend Snippet: Sirt1 overexpression or activation dampens Aicda expression and CSR, while Sirt1 deletion further increases Aicda expression and CSR. ( A ) Ex vivo expression of Sirt1 and Aicda (qRT-PCR) in peripheral blood total B cells isolated from non-intentionally immunized Sirt1 +/+ and Sirt1 super mice. Data are ratios to expression in total B cells from Sirt1 +/+ mice (set as 1; means ± SEM of values from three Sirt1 +/+ mice and three Sirt1 super mice). ( B ) Serum titers [enzyme-linked immunosorbent assay (ELISA)] of total IgM, IgG1, and IgA in non-intentionally immunized Sirt1 +/+ and Sirt1 super mice (means ± SD of six Sirt1 +/+ mice and six Sirt1 super mice). ( C ) Expression of Sirt1, Aicda, Prdm1 , and Xbp1 transcripts (qRT-PCR) in Sirt1 +/+ (set as 1) and Sirt1 super B cells stimulated with LPS plus IL-4 for 72 hours (means ± SEM of three biological independent experiments, each consisting of triplicates; left) as well as immunoblotting and flow cytometry analysis of Sirt1 and AID protein levels in these cells (one of two independent experiments yielding similar results; right). ( D and E ) CSR (D) and plasma cell differentiation (E) in Sirt1 +/+ and Sirt1 super B cells (flow cytometry analysis) after stimulation with appropriate stimuli, as indicated, for 96 hours (left, one representative of three independent experiments; right, means ± SEM of three independent experiments). ( F ) CSR to IgG1 (flow cytometry analysis) in (B220 + GFP + ) B cells transduced by pMIG-GFP or pMIG-GFP-Sirt1 retrovirus (one of two independent experiments yielding similar results). ( G to I ) C57BL/6 B cells stimulated for 96 hours with LPS plus IL-4 in the presence of SRT1720 at indicated doses. Percentages of IgG1 + B cells and B220 low CD138 + plasma cells (G), cell proliferation [carboxyfluorescein diacetate succinimidyl ester (CFSE)–labeled cells] (H), and viability [7-aminoactinomycin D–negative (7-AAD − )] (I) (flow cytometry analysis). Data are means ± SEM of three independent experiments (G) or one representative of three independent experiments (H and I). ( J ) Aicda, Prdm1 , and Xbp1 transcript levels (qRT-PCR analysis) in purified B cells treated with nil or SRT1720 at the indicated doses and stimulated for 72 hours with LPS plus IL-4. Data are ratios to the expression in B cells treated with nil (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). ( K ) Sirt1 transcript levels (qRT-PCR analysis) in Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl B cells stimulated with LPS plus IL-4 for 0 and 72 hours. Data are ratios to the expression in unstimulated (0 hours) Aicda cre Sirt1 +/+ B cells (set as 1; ± SEM of three biological independent experiments, each consisting of triplicates). ( L ) CSR to different Ig isotypes (flow cytometry analysis) in Aidca cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl B cells stimulated for 96 hours with appropriate stimuli, as indicated. Data are one representative (left) and means ± SEM (right) of three independent experiments. ( M ) Expression of indicated genes (qRT-PCR analysis) in Aidca cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl B cells stimulated for 72 hours with LPS plus IL-4 or LPS plus TGF-β and anti-Igδ mAb-dextran (Iα-Cα and Iμ-Cα). Data are ratios to the expression in Aicda cre Sirt1 +/+ B cells (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). ( N ) AID protein levels in Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl B cells stimulated with LPS plus IL-4 for 72 hours as analyzed by intracellular staining and flow cytometry. Data are from one of two independent experiments yielding similar results. ( O ) Plasma cell differentiation (flow cytometry analysis) in Aidca cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl B cells stimulated for 96 hours with appropriate stimuli, as indicated. Data are one representative (left) and means ± SEM (right) of three biological independent experiments. ( P ) Proliferation of spleen B220 + B cells labeled with CFSE and stimulated with LPS plus IL-4 for 72 and 96 hours (top) and B cell viability (7-AAD − , bottom). Data are one representative of three independent experiments yielding similar results. * P

Techniques Used: Over Expression, Activation Assay, Expressing, Ex Vivo, Quantitative RT-PCR, Isolation, Mouse Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cell Differentiation, Labeling, Purification, Staining

B cell Sirt1 reduction or ablation enhances acetylation of Aicda promoter histones, Dnmt1, and NF-κB p65, while overexpression or activation of B cell Sirt1 results in reduced acetylation of these proteins. ( A and B ) ChIP-qPCR analysis of acetylated H3K9/K14 in Aicda, Prdm1 , and Xbp1 promoters (A) and Sirt1 recruitment to the Aicda promoter (B) of WT C57BL/6 B cells nonstimulated (Nil) or stimulated with LPS plus IL-4 for 72 hours. Data are ratios to nonstimulated B cells (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). ( C to I ) Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl B cells were stimulated with LPS plus IL-4 (C to I) or CD154 plus IL-4 (G to I) for 72 hours. (C) Acetylated Dnmt1 and total Dnmt1 (left, immunoblotting; one representative of three independent experiments yielding similar results) and densitometry quantification of signals normalized to β-actin levels and depicted as ratios to signals in Aicda cre Sirt1 +/+ B cells (set as 1; means ± SD of three independent experiments; right). (D) Recruitment of Dnmt1 to the Aicda promoter or a Prdm1 regulatory region (ChIP-qPCR). Data are ratios to recruitment in Aicda cre Sirt1 fl/fl B cells to Aicda cre Sirt1 +/+ B cells (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). (E) CpG DNA methylation at the Aicda promoter and Prdm1 regulatory region, as assessed by bisulfite sequencing. Depicted is the proportion of methylated dC nucleotides within the CpG motifs (pooled data from two mice in each group, with at least 5000 sequences from each mouse; means ± SD). (F) DNA methylation of Aicda and Xbp1 promoters, as well as Prdm1 regulatory region (MeDIP-qPCR). Data are normalized to DNA used as input (means ± SEM of three biological independent experiments, each consisting of triplicates). (G and H) Immunoblotting analysis of acetylated NF-κB p65 and total NF-κB p65 (G) and densitometry quantification of signals normalized to β-actin levels and depicted as ratios to signals in Aicda cre Sirt1 +/+ B cells (set as 1; means ± SD of three independent experiments) (H). (I) Recruitment of acetylated NF-κB p65 to Aicda promoter or Prdm1 regulatory region (ChIP-qPCR) in B cells. Data are ratios of recruitment in Aicda cre Sirt1 fl/fl cells to that in Aicda cre Sirt1 +/+ B cells (set as 1; means ± SEM of three biological independent experiments). ( J to L ) Sirt1 +/+ and Sirt1 super B cells were stimulated with LPS plus IL-4 for 72 hours. (J) Acetylated H3K9/K14 in the Aicda, Prdm1 , or Xbp1 promoter (ChIP-qPCR). Data are ratios of determinations in Sirt1 super B cells to those in Sirt1 +/+ B cells (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). (K) Acetylated NF-κB p65 and total NF-κB p65 (immunoblotting analysis; left) and densitometry quantification of signals normalized to β-actin levels and depicted as the ratio to that in Sirt1 +/+ B cells (set as 1; means ± SD of three independent experiments; right). (L) Recruitment of acetylated NF-κB p65 to Aicda, Prdm1 , and Xbp1 promoters (ChIP-qPCR analysis). Data are ratios to recruitment in Sirt1 +/+ B cells (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). ( M ) Aicda expression (qRT-PCR analysis) in Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl B cells treated with nil or SRT1720 and stimulated with LPS plus IL-4 for 72 hours (means ± SEM of three biological independent experiments, each consisting of triplicates). ( N to Q ) WT C57BL/6 B cells were treated with nil or SRT1720 and stimulated with LPS plus IL-4 for 72 hours. (N and O) ChIP-qPCR analysis of Sirt1 recruitment to Aicda, Prdm1 , and Xbp1 promoters (N) as well as acetylated H3K9/K14 of these regions (O). Data are ratios of readings in SRT1720-treated B cells to those in nil-treated B cells (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). (P) Immunoblotting of acetylated NF-κB p65 and total NF-κB p65 in B cells; densitometry quantification of signals normalized to β-actin levels and depicted as ratios of readings in SRT1720-treated B cells to those in nil-treated B cells (set as 1; means ± SD of three independent experiments). (Q) Recruitment of acetylated NF-κB p65 to the Aicda, Prdm1 , and Xbp1 promoters (ChIP-qPCR analysis). Data are ratios of readings in SRT1720-treated B cells to those in nil-treated B cells (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). * P
Figure Legend Snippet: B cell Sirt1 reduction or ablation enhances acetylation of Aicda promoter histones, Dnmt1, and NF-κB p65, while overexpression or activation of B cell Sirt1 results in reduced acetylation of these proteins. ( A and B ) ChIP-qPCR analysis of acetylated H3K9/K14 in Aicda, Prdm1 , and Xbp1 promoters (A) and Sirt1 recruitment to the Aicda promoter (B) of WT C57BL/6 B cells nonstimulated (Nil) or stimulated with LPS plus IL-4 for 72 hours. Data are ratios to nonstimulated B cells (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). ( C to I ) Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl B cells were stimulated with LPS plus IL-4 (C to I) or CD154 plus IL-4 (G to I) for 72 hours. (C) Acetylated Dnmt1 and total Dnmt1 (left, immunoblotting; one representative of three independent experiments yielding similar results) and densitometry quantification of signals normalized to β-actin levels and depicted as ratios to signals in Aicda cre Sirt1 +/+ B cells (set as 1; means ± SD of three independent experiments; right). (D) Recruitment of Dnmt1 to the Aicda promoter or a Prdm1 regulatory region (ChIP-qPCR). Data are ratios to recruitment in Aicda cre Sirt1 fl/fl B cells to Aicda cre Sirt1 +/+ B cells (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). (E) CpG DNA methylation at the Aicda promoter and Prdm1 regulatory region, as assessed by bisulfite sequencing. Depicted is the proportion of methylated dC nucleotides within the CpG motifs (pooled data from two mice in each group, with at least 5000 sequences from each mouse; means ± SD). (F) DNA methylation of Aicda and Xbp1 promoters, as well as Prdm1 regulatory region (MeDIP-qPCR). Data are normalized to DNA used as input (means ± SEM of three biological independent experiments, each consisting of triplicates). (G and H) Immunoblotting analysis of acetylated NF-κB p65 and total NF-κB p65 (G) and densitometry quantification of signals normalized to β-actin levels and depicted as ratios to signals in Aicda cre Sirt1 +/+ B cells (set as 1; means ± SD of three independent experiments) (H). (I) Recruitment of acetylated NF-κB p65 to Aicda promoter or Prdm1 regulatory region (ChIP-qPCR) in B cells. Data are ratios of recruitment in Aicda cre Sirt1 fl/fl cells to that in Aicda cre Sirt1 +/+ B cells (set as 1; means ± SEM of three biological independent experiments). ( J to L ) Sirt1 +/+ and Sirt1 super B cells were stimulated with LPS plus IL-4 for 72 hours. (J) Acetylated H3K9/K14 in the Aicda, Prdm1 , or Xbp1 promoter (ChIP-qPCR). Data are ratios of determinations in Sirt1 super B cells to those in Sirt1 +/+ B cells (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). (K) Acetylated NF-κB p65 and total NF-κB p65 (immunoblotting analysis; left) and densitometry quantification of signals normalized to β-actin levels and depicted as the ratio to that in Sirt1 +/+ B cells (set as 1; means ± SD of three independent experiments; right). (L) Recruitment of acetylated NF-κB p65 to Aicda, Prdm1 , and Xbp1 promoters (ChIP-qPCR analysis). Data are ratios to recruitment in Sirt1 +/+ B cells (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). ( M ) Aicda expression (qRT-PCR analysis) in Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl B cells treated with nil or SRT1720 and stimulated with LPS plus IL-4 for 72 hours (means ± SEM of three biological independent experiments, each consisting of triplicates). ( N to Q ) WT C57BL/6 B cells were treated with nil or SRT1720 and stimulated with LPS plus IL-4 for 72 hours. (N and O) ChIP-qPCR analysis of Sirt1 recruitment to Aicda, Prdm1 , and Xbp1 promoters (N) as well as acetylated H3K9/K14 of these regions (O). Data are ratios of readings in SRT1720-treated B cells to those in nil-treated B cells (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). (P) Immunoblotting of acetylated NF-κB p65 and total NF-κB p65 in B cells; densitometry quantification of signals normalized to β-actin levels and depicted as ratios of readings in SRT1720-treated B cells to those in nil-treated B cells (set as 1; means ± SD of three independent experiments). (Q) Recruitment of acetylated NF-κB p65 to the Aicda, Prdm1 , and Xbp1 promoters (ChIP-qPCR analysis). Data are ratios of readings in SRT1720-treated B cells to those in nil-treated B cells (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). * P

Techniques Used: Over Expression, Activation Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, DNA Methylation Assay, Methylation Sequencing, Methylation, Mouse Assay, Methylated DNA Immunoprecipitation, Expressing, Quantitative RT-PCR

Increased glucose concentration reduces cytosolic NAD + , increases acetylation of Aicda promoter histones, Dnmt1 and NF-κB p65, and enhances Aicda expression and CSR. B cells were cultured in glucose-free fetal bovine serum (FBS)–RPMI medium supplemented with increased concentrations of ( A ) glucose or ( B ) galactose or in complete FBS-RPMI-1640 medium supplemented with increased concentrations of ( C ) 2-DG or ( D ) NAD + and stimulated with LPS plus IL-4. Surface expression of B220 and IgG1 was analyzed by flow cytometry after 96 hours of culture. Expression of Aicda, Prdm1 , and Irf4 was analyzed by qRT-PCR after 72 hours of culture. NAD + and NADH concentrations in B cells cultured with increased amount of glucose were also determined after 72 hours. Data are from one representative of three independent experiments yielding comparable results (left) or means ± SEM of three biological independent experiments, each consisting of triplicates (right). C57BL/6 ( E to L ) or Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl ( M and N ) B cells were cultured in glucose-free FBS-RPMI medium supplied with indicated concentrations of glucose and stimulated with LPS plus IL-4 for 72 (E to M) or 96 hours (N). (E and F) ChIP-qPCR analysis of acetylated H3K9/K14 in Aicda, Prdm1 , and Xbp1 promoters (E) and recruitment of Sirt1 to the Aicda promoter (F) in B cells cultured with 0 or 20 mM of glucose. Data are ratios to recruitment in nil-treated B cells (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). (G) Densitometry quantification of immunoblotting signals of acetylated Dnmt1 and total Dnmt1 after normalization to β-actin levels in B cells. Data are ratios of acetylated Dnmt1 and Dnmt1 in B cells cultured with increased concentrations of glucose to B cells cultured without glucose (set as 1; means ± SD of three independent experiments). (H) ChIP-qPCR analysis of recruitment of Dnmt1 to the Aicda promoter in B cells cultured in different concentrations of glucose, as indicated. Data are ratios of recruitment in B cells cultured with glucose to that in B cells cultured without glucose (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). (I) MeDIP-qPCR analysis of DNA methylation in the Aicda promoter of B cells cultured in different concentrations of glucose, as indicated. Data are ratios of values in B cells cultured in glucose to those in B cells cultured without glucose (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). (J and K) Immunoblotting analysis of acetylated NF-κB p65 and total NF-κB p65 in B cells cultured in different concentrations of glucose, as indicated (J); densitometry quantification of signals expressed as ratios of signal in B cells cultured in glucose to that in B cells cultured without glucose (set as 1; means ± SD of three independent experiments) (K). (L) Recruitment of acetylated NF-κB p65 to the Aicda promoter (ChIP-qPCR analysis) in induced B cells. Data are ratios of recruitment of acetylated NF-κB p65 to Aicda promoter in B cells cultured with glucose to that in B cells cultured without glucose (set as 1; means ± SEM of three biological independent experiments). (M) qRT-PCR analysis of Aicda expression in Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl B cells cultured in the different concentrations of glucose. Data are ratios of Aicda expression in Aicda cre Sirt1 fl/fl B cells to that in Aicda cre Sirt1 +/+ B cells (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). (N) Flow cytometry analysis of CSR to IgG1 in Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl B cells cultured in different concentrations of glucose. Data are ratios of IgG1 + Aicda cre Sirt1 fl/fl B cells to Aicda cre Sirt1 +/+ B cells (set as 1 in each glucose concentration; means ± SEM of three biological independent experiments). * P
Figure Legend Snippet: Increased glucose concentration reduces cytosolic NAD + , increases acetylation of Aicda promoter histones, Dnmt1 and NF-κB p65, and enhances Aicda expression and CSR. B cells were cultured in glucose-free fetal bovine serum (FBS)–RPMI medium supplemented with increased concentrations of ( A ) glucose or ( B ) galactose or in complete FBS-RPMI-1640 medium supplemented with increased concentrations of ( C ) 2-DG or ( D ) NAD + and stimulated with LPS plus IL-4. Surface expression of B220 and IgG1 was analyzed by flow cytometry after 96 hours of culture. Expression of Aicda, Prdm1 , and Irf4 was analyzed by qRT-PCR after 72 hours of culture. NAD + and NADH concentrations in B cells cultured with increased amount of glucose were also determined after 72 hours. Data are from one representative of three independent experiments yielding comparable results (left) or means ± SEM of three biological independent experiments, each consisting of triplicates (right). C57BL/6 ( E to L ) or Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl ( M and N ) B cells were cultured in glucose-free FBS-RPMI medium supplied with indicated concentrations of glucose and stimulated with LPS plus IL-4 for 72 (E to M) or 96 hours (N). (E and F) ChIP-qPCR analysis of acetylated H3K9/K14 in Aicda, Prdm1 , and Xbp1 promoters (E) and recruitment of Sirt1 to the Aicda promoter (F) in B cells cultured with 0 or 20 mM of glucose. Data are ratios to recruitment in nil-treated B cells (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). (G) Densitometry quantification of immunoblotting signals of acetylated Dnmt1 and total Dnmt1 after normalization to β-actin levels in B cells. Data are ratios of acetylated Dnmt1 and Dnmt1 in B cells cultured with increased concentrations of glucose to B cells cultured without glucose (set as 1; means ± SD of three independent experiments). (H) ChIP-qPCR analysis of recruitment of Dnmt1 to the Aicda promoter in B cells cultured in different concentrations of glucose, as indicated. Data are ratios of recruitment in B cells cultured with glucose to that in B cells cultured without glucose (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). (I) MeDIP-qPCR analysis of DNA methylation in the Aicda promoter of B cells cultured in different concentrations of glucose, as indicated. Data are ratios of values in B cells cultured in glucose to those in B cells cultured without glucose (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). (J and K) Immunoblotting analysis of acetylated NF-κB p65 and total NF-κB p65 in B cells cultured in different concentrations of glucose, as indicated (J); densitometry quantification of signals expressed as ratios of signal in B cells cultured in glucose to that in B cells cultured without glucose (set as 1; means ± SD of three independent experiments) (K). (L) Recruitment of acetylated NF-κB p65 to the Aicda promoter (ChIP-qPCR analysis) in induced B cells. Data are ratios of recruitment of acetylated NF-κB p65 to Aicda promoter in B cells cultured with glucose to that in B cells cultured without glucose (set as 1; means ± SEM of three biological independent experiments). (M) qRT-PCR analysis of Aicda expression in Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl B cells cultured in the different concentrations of glucose. Data are ratios of Aicda expression in Aicda cre Sirt1 fl/fl B cells to that in Aicda cre Sirt1 +/+ B cells (set as 1; means ± SEM of three biological independent experiments, each consisting of triplicates). (N) Flow cytometry analysis of CSR to IgG1 in Aicda cre Sirt1 +/+ and Aicda cre Sirt1 fl/fl B cells cultured in different concentrations of glucose. Data are ratios of IgG1 + Aicda cre Sirt1 fl/fl B cells to Aicda cre Sirt1 +/+ B cells (set as 1 in each glucose concentration; means ± SEM of three biological independent experiments). * P

Techniques Used: Concentration Assay, Expressing, Cell Culture, Flow Cytometry, Quantitative RT-PCR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Methylated DNA Immunoprecipitation, DNA Methylation Assay

24) Product Images from "IL-4 and IL-13 Compromise the Sinonasal Epithelial Barrier and Perturb Intercellular Junction Protein Expression"

Article Title: IL-4 and IL-13 Compromise the Sinonasal Epithelial Barrier and Perturb Intercellular Junction Protein Expression

Journal: International forum of allergy & rhinology

doi: 10.1002/alr.21298

(a) Graph of pooled Western blot densitometry values across in vitro sinonasal epithelial cytokine exposure experiments. Pooled densitometry values for JAM-A and E-cadherin reveal a decrease following IL-4 and IL-13 exposure (*), whereas densitometry
Figure Legend Snippet: (a) Graph of pooled Western blot densitometry values across in vitro sinonasal epithelial cytokine exposure experiments. Pooled densitometry values for JAM-A and E-cadherin reveal a decrease following IL-4 and IL-13 exposure (*), whereas densitometry

Techniques Used: Western Blot, In Vitro

25) Product Images from "The Low Affinity IgE Receptor (CD23) Is Cleaved by the Metalloproteinase ADAM10 *"

Article Title: The Low Affinity IgE Receptor (CD23) Is Cleaved by the Metalloproteinase ADAM10 *

Journal:

doi: 10.1074/jbc.M608414200

Influence of IL-4 on CD23 and ADAM10 expression
Figure Legend Snippet: Influence of IL-4 on CD23 and ADAM10 expression

Techniques Used: Expressing

26) Product Images from "A stabilized HIV-1 envelope glycoprotein trimer fused to CD40 ligand targets and activates dendritic cells"

Article Title: A stabilized HIV-1 envelope glycoprotein trimer fused to CD40 ligand targets and activates dendritic cells

Journal: Retrovirology

doi: 10.1186/1742-4690-8-48

SOSIP.R6-IZ-CD40L-exposed DC prime naïve CD4 + T-cells . A . HLA-DR expression on CD4+ T-cells cultured for 5 days in the absence or presence of DC that were exposed to SOSIP.R6-IZ, SOSIP.R6-IZ-CD40L, or control stimuli was measured by FACS. Percentages of CD3 + HLA-DR + T-cells are displayed within each graph. Relative number of T cells positive for cell surface expression of CD45RO (the percentages of positive cells are given in the text) (B) , and intracellular expression of IFN-γ (C) and IL-4 (D) . IL-2 (E) , IFN-γ (F) and IL-4 (G) levels in the supernatant of DC-T cell co-cultures at day 5 measured by ELISA. Data are representative for three independent experiments.
Figure Legend Snippet: SOSIP.R6-IZ-CD40L-exposed DC prime naïve CD4 + T-cells . A . HLA-DR expression on CD4+ T-cells cultured for 5 days in the absence or presence of DC that were exposed to SOSIP.R6-IZ, SOSIP.R6-IZ-CD40L, or control stimuli was measured by FACS. Percentages of CD3 + HLA-DR + T-cells are displayed within each graph. Relative number of T cells positive for cell surface expression of CD45RO (the percentages of positive cells are given in the text) (B) , and intracellular expression of IFN-γ (C) and IL-4 (D) . IL-2 (E) , IFN-γ (F) and IL-4 (G) levels in the supernatant of DC-T cell co-cultures at day 5 measured by ELISA. Data are representative for three independent experiments.

Techniques Used: Expressing, Cell Culture, FACS, Enzyme-linked Immunosorbent Assay

27) Product Images from "Fluticasone propionate-induced regulation of the balance within macrophage subpopulations"

Article Title: Fluticasone propionate-induced regulation of the balance within macrophage subpopulations

Journal: Clinical and Experimental Immunology

doi: 10.1046/j.1365-2249.2000.01085.x

Effect of fluticasone propionate (FP) and IL-4 in combination on macrophage phenotype. The proportions of monocytes expressing the stimulatory phenotype D1 + ; phagocytic phenotype D7 + ; and suppressive phenotype D1/D7 + after 7 days culture in the absence or presence of FP and/or IL-4 added on day 5. Experiment performed in duplicate. Results of typical experiment are shown.
Figure Legend Snippet: Effect of fluticasone propionate (FP) and IL-4 in combination on macrophage phenotype. The proportions of monocytes expressing the stimulatory phenotype D1 + ; phagocytic phenotype D7 + ; and suppressive phenotype D1/D7 + after 7 days culture in the absence or presence of FP and/or IL-4 added on day 5. Experiment performed in duplicate. Results of typical experiment are shown.

Techniques Used: Expressing

Effect on steroid-treated monocytes on T cell proliferation. The effect of corticosteroids and IL-4 on T cell proliferation in an allogeneic mixed leucocyte reaction (MLR). The results are expressed as ct/min of 3 H-thymidine. Experiment performed three times, bars represent mean ± s.e.m. of stimulation index (SI). (Ct/min for allogeneic reactivity without cytokine addition were reduced to unity and all other results are represented as a SI in relation to this result.)
Figure Legend Snippet: Effect on steroid-treated monocytes on T cell proliferation. The effect of corticosteroids and IL-4 on T cell proliferation in an allogeneic mixed leucocyte reaction (MLR). The results are expressed as ct/min of 3 H-thymidine. Experiment performed three times, bars represent mean ± s.e.m. of stimulation index (SI). (Ct/min for allogeneic reactivity without cytokine addition were reduced to unity and all other results are represented as a SI in relation to this result.)

Techniques Used:

28) Product Images from "Uncarinic Acid C Isolated from Uncaria rhynchophylla Induces Differentiation of Th1-Promoting Dendritic Cells Through TLR4 Signaling"

Article Title: Uncarinic Acid C Isolated from Uncaria rhynchophylla Induces Differentiation of Th1-Promoting Dendritic Cells Through TLR4 Signaling

Journal: Biomarker Insights

doi: 10.4137/BMI.S6441

Immature DC stimulated with URC (0.1 μM)-primed DC, LPS-prime DC or TNF-α-primed DC induced Th1-polarizing capacity. Allogeneic DC were co-cultured with naïve T cells in the presence of control Ab or anti-IL-12 mAb (10 μg/ml). After 9 days of expansion in IL-2, T cells were counted and re-stimulated with CD3/CD28 for 24 h. As a control, immature DC were used. IFN-γ ( A ) and IL-4 ( B ) were measured by ELISA in culture supernatants. Data are the mean ± S.E.M. of five independent experiments. Note: * P
Figure Legend Snippet: Immature DC stimulated with URC (0.1 μM)-primed DC, LPS-prime DC or TNF-α-primed DC induced Th1-polarizing capacity. Allogeneic DC were co-cultured with naïve T cells in the presence of control Ab or anti-IL-12 mAb (10 μg/ml). After 9 days of expansion in IL-2, T cells were counted and re-stimulated with CD3/CD28 for 24 h. As a control, immature DC were used. IFN-γ ( A ) and IL-4 ( B ) were measured by ELISA in culture supernatants. Data are the mean ± S.E.M. of five independent experiments. Note: * P

Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay

URC induces NF-κB activation and modulates TLR2 and TLR4. A ) DC were stimulated with URC (0.5 μM), LPS (100 ng/ml) or medium for 30 min, 1 h and 2 h, and nuclear extracts were analyzed for their NF-κB binding activity using EMSA. 1: Competition (×100 cold) negative control 2: immature DC (0 min), 3: immature DC (30 min) 4: immature DC (1 h) 5: immature DC (2 h) 6: LPS (0 min) 7: LPS (30 min) 8: LPS (1 h) 9: LPS (2 h) 10: URC (0 min) 11: URC (30 min) 12: URC (1 h) 13: URC (2 h). B ) Relative TLR expression during the differentiation of DC. Monocytes were cultured with GM-CSF and IL-4 to differentiate to immature DC for 8 days. Maturation of DC was induced by URC (0.1 μM and 0.5 μM) or LPS (1 μg/ml). Cells were collected at indicated time; mRNA were extracted and converted to cDNA. The cDNA were subjected to Realtime SYBR Green quantitative PCR using gene-specific primers pair for TLR2, TLR4 and β-actin. Relative gene expression was calculated using 2 −ΔΔCt method. Left panel: Relative TLR4 expression during differentiation of DC by LPS and comparison calculation involved finding the stimulation of DC between LPS and URC. Right panel: Relative TLR2 expression during differentiation of DC by LPS and comparison calculation involved finding the stimulation of DC between LPS and URC. Data are the mean ± S.E.M. of three independent experiments. Note: * P
Figure Legend Snippet: URC induces NF-κB activation and modulates TLR2 and TLR4. A ) DC were stimulated with URC (0.5 μM), LPS (100 ng/ml) or medium for 30 min, 1 h and 2 h, and nuclear extracts were analyzed for their NF-κB binding activity using EMSA. 1: Competition (×100 cold) negative control 2: immature DC (0 min), 3: immature DC (30 min) 4: immature DC (1 h) 5: immature DC (2 h) 6: LPS (0 min) 7: LPS (30 min) 8: LPS (1 h) 9: LPS (2 h) 10: URC (0 min) 11: URC (30 min) 12: URC (1 h) 13: URC (2 h). B ) Relative TLR expression during the differentiation of DC. Monocytes were cultured with GM-CSF and IL-4 to differentiate to immature DC for 8 days. Maturation of DC was induced by URC (0.1 μM and 0.5 μM) or LPS (1 μg/ml). Cells were collected at indicated time; mRNA were extracted and converted to cDNA. The cDNA were subjected to Realtime SYBR Green quantitative PCR using gene-specific primers pair for TLR2, TLR4 and β-actin. Relative gene expression was calculated using 2 −ΔΔCt method. Left panel: Relative TLR4 expression during differentiation of DC by LPS and comparison calculation involved finding the stimulation of DC between LPS and URC. Right panel: Relative TLR2 expression during differentiation of DC by LPS and comparison calculation involved finding the stimulation of DC between LPS and URC. Data are the mean ± S.E.M. of three independent experiments. Note: * P

Techniques Used: Activation Assay, Binding Assay, Activity Assay, Negative Control, Expressing, Cell Culture, SYBR Green Assay, Real-time Polymerase Chain Reaction

29) Product Images from "Fingerprints of CD8+ T cells on human pre-plasma and memory B cells"

Article Title: Fingerprints of CD8+ T cells on human pre-plasma and memory B cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0208187

CD8 positive B cells release cytokines and immunoglobulins. (A) Production of the cytokines EBI3 and IL-35 was determined by ELISA from supernatants of freshly sorted CD8+ B cells (black bars) and CD8- control B cells (white bars). Cultures contained 50’000 cells in 200 μl volume. As indicated, samples for both measurements were obtained at 18h and 4 days, without or with CD40+IL-4 stimulation. The dashed line indicates the assay detection limit. (B) Production of IgG was determined by ELISPOT using cells sorted either from apheresis material or from peripheral blood (PBMC). Spots were counted, and are expressed as percentage of input cells (3’000 or 1’000 cells per well, as indicated). Cultures were performed without (black bars) or with CD40L+IL-4 stimulation (white bars).
Figure Legend Snippet: CD8 positive B cells release cytokines and immunoglobulins. (A) Production of the cytokines EBI3 and IL-35 was determined by ELISA from supernatants of freshly sorted CD8+ B cells (black bars) and CD8- control B cells (white bars). Cultures contained 50’000 cells in 200 μl volume. As indicated, samples for both measurements were obtained at 18h and 4 days, without or with CD40+IL-4 stimulation. The dashed line indicates the assay detection limit. (B) Production of IgG was determined by ELISPOT using cells sorted either from apheresis material or from peripheral blood (PBMC). Spots were counted, and are expressed as percentage of input cells (3’000 or 1’000 cells per well, as indicated). Cultures were performed without (black bars) or with CD40L+IL-4 stimulation (white bars).

Techniques Used: Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

30) Product Images from "Suppressive Effect of Quercetin on Nitric Oxide Production from Nasal Epithelial Cells In Vitro"

Article Title: Suppressive Effect of Quercetin on Nitric Oxide Production from Nasal Epithelial Cells In Vitro

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

doi: 10.1155/2018/6097625

Influence of leflunomide on IL-4-induced NO production from HNEpCs in vitro. HNEpCs at 1 x 10 5 cells/ml are cultured with 10.0 ng/ml of IL-4 for 48 hours in the presence and absence of various concentrations of leflunomide. The NO levels in the culture supernatants are measured using the Griess method, and the results are expressed as the mean μ M ± SE of the triplicate cultures. The experiments are performed twice with similar results. Med. alone: medium alone; IL-4: interleukin-4; NO: nitric oxide; HNEpCs: human nasal epithelial cells.
Figure Legend Snippet: Influence of leflunomide on IL-4-induced NO production from HNEpCs in vitro. HNEpCs at 1 x 10 5 cells/ml are cultured with 10.0 ng/ml of IL-4 for 48 hours in the presence and absence of various concentrations of leflunomide. The NO levels in the culture supernatants are measured using the Griess method, and the results are expressed as the mean μ M ± SE of the triplicate cultures. The experiments are performed twice with similar results. Med. alone: medium alone; IL-4: interleukin-4; NO: nitric oxide; HNEpCs: human nasal epithelial cells.

Techniques Used: In Vitro, Cell Culture

Influence of quercetin on STAT6 activation in HNEpCs after IL-4 stimulation in vitro . HNEpCs at 1 x 10 5 cells/ml are cultured with 10.0 ng/ml of IL-4 for 12 hours in the presence and absence of various concentrations of quercetin. STAT6 activation is measured using ELISA, and the results are expressed as the mean optical density at 450 nm ± SE of the triplicate cultures. The experiments are performed twice with similar results. Med. alone: medium alone; IL-4: interleukin-4; HNEpCs: human nasal epithelial cells.
Figure Legend Snippet: Influence of quercetin on STAT6 activation in HNEpCs after IL-4 stimulation in vitro . HNEpCs at 1 x 10 5 cells/ml are cultured with 10.0 ng/ml of IL-4 for 12 hours in the presence and absence of various concentrations of quercetin. STAT6 activation is measured using ELISA, and the results are expressed as the mean optical density at 450 nm ± SE of the triplicate cultures. The experiments are performed twice with similar results. Med. alone: medium alone; IL-4: interleukin-4; HNEpCs: human nasal epithelial cells.

Techniques Used: Activation Assay, In Vitro, Cell Culture, Enzyme-linked Immunosorbent Assay

Influence of quercetin on NO production from HNEpCs after IL-4 stimulation in vitro . HNEpCs at 1 x 10 5 cells/ml are cultured with 10.0 ng/ml of IL-4 for 48 hours in the presence and absence of various concentrations of quercetin. The NO levels in the culture supernatants are measured using the Griess method, and the results are expressed as the mean μ M ± SE of the triplicate cultures. The experiments are performed twice with similar results. Med. alone: medium alone; IL-4: interleukin-4; NO: nitric oxide; HNEpCs: human nasal epithelial cells.
Figure Legend Snippet: Influence of quercetin on NO production from HNEpCs after IL-4 stimulation in vitro . HNEpCs at 1 x 10 5 cells/ml are cultured with 10.0 ng/ml of IL-4 for 48 hours in the presence and absence of various concentrations of quercetin. The NO levels in the culture supernatants are measured using the Griess method, and the results are expressed as the mean μ M ± SE of the triplicate cultures. The experiments are performed twice with similar results. Med. alone: medium alone; IL-4: interleukin-4; NO: nitric oxide; HNEpCs: human nasal epithelial cells.

Techniques Used: In Vitro, Cell Culture

Influence of IL-4 stimulation on NO production from HNEpCs in vitro . HNEpCs at 1 x 10 5 cells/ml are stimulated with different concentrations of IL-4 for 24 to 72 hours. NO levels in the culture supernatants are measured using the Griess method, and the results are expressed as the mean μ M ± SE of the triplicate cultures. (a) Dose-response profile of IL-4 on NO production; (b) time course of IL-4-induced NO production. The experiments are performed twice with similar results. Med. alone: medium alone; IL-4: interleukin-4; NO: nitric oxide; HNEpCs: human nasal epithelial cells.
Figure Legend Snippet: Influence of IL-4 stimulation on NO production from HNEpCs in vitro . HNEpCs at 1 x 10 5 cells/ml are stimulated with different concentrations of IL-4 for 24 to 72 hours. NO levels in the culture supernatants are measured using the Griess method, and the results are expressed as the mean μ M ± SE of the triplicate cultures. (a) Dose-response profile of IL-4 on NO production; (b) time course of IL-4-induced NO production. The experiments are performed twice with similar results. Med. alone: medium alone; IL-4: interleukin-4; NO: nitric oxide; HNEpCs: human nasal epithelial cells.

Techniques Used: In Vitro

Influence of quercetin on iNOS mRNA expression in HNEpCs after IL-4 stimulation in vitro . HNEpCs at 1 x 10 5 cells/ml are cultured with 10.0 ng/ml of IL-4 for 24 hours in the presence and absence of various concentrations of quercetin. iNOS mRNA expression is measured using quantitative real-time RT-PCR, and the results are expressed as the mean RQ ± SE of the triplicate cultures. The experiments are performed twice with similar results. Med. alone: medium alone; IL-4: interleukin-4; HNEpCs: human nasal epithelial cells; iNOS: inducible nitric oxide synthase; RQ: relative quantification.
Figure Legend Snippet: Influence of quercetin on iNOS mRNA expression in HNEpCs after IL-4 stimulation in vitro . HNEpCs at 1 x 10 5 cells/ml are cultured with 10.0 ng/ml of IL-4 for 24 hours in the presence and absence of various concentrations of quercetin. iNOS mRNA expression is measured using quantitative real-time RT-PCR, and the results are expressed as the mean RQ ± SE of the triplicate cultures. The experiments are performed twice with similar results. Med. alone: medium alone; IL-4: interleukin-4; HNEpCs: human nasal epithelial cells; iNOS: inducible nitric oxide synthase; RQ: relative quantification.

Techniques Used: Expressing, In Vitro, Cell Culture, Quantitative RT-PCR

31) Product Images from "Activated Natural Killer Cells Mediate the Suppressive Effect of Interleukin-4 on Tumor Development via STAT6 Activation in an Atopic Condition Melanoma Model"

Article Title: Activated Natural Killer Cells Mediate the Suppressive Effect of Interleukin-4 on Tumor Development via STAT6 Activation in an Atopic Condition Melanoma Model

Journal: Neoplasia (New York, N.Y.)

doi: 10.1016/j.neo.2017.02.014

Allergy responses inhibited IL-4–mediated tumor growth in a melanoma model. (A) Tumor growth inhibition (as assessed by tumor weight and volume) in B16F10-bearing PA-treated IL-4 mice and AOO-treated IL-4 control mice. The tumor burden was measured twice per week using a caliper, and the volume was calculated according to the following formula: volume length (mm) × width (mm) × height (mm)/2. Tumor weight and volume are presented as means ± SD from eight mice. (B) Immunohistochemistry was used to determine the expression levels of proliferating cell nuclear antigen in the melanoma tissues of PA-treated IL-4 mice and AOO-treated IL-4 mice. (C) The expression of IL-4 and apoptotic proteins was detected by Western blotting using specific antibodies: IL-4, IL-4Rα, JAK1, p-JAK1, cleaved caspase-3, -8, -9, BCL-2, and β-actin; β-actin was used as an internal control. (D) STAT6 activity in tumor tissues was detected by EMSA, and (E) the nuclear location of STAT6 was determined by Western blotting; histone protein was used as an internal control. All values represent the mean ± SD from eight animal tumor sections. * ( P
Figure Legend Snippet: Allergy responses inhibited IL-4–mediated tumor growth in a melanoma model. (A) Tumor growth inhibition (as assessed by tumor weight and volume) in B16F10-bearing PA-treated IL-4 mice and AOO-treated IL-4 control mice. The tumor burden was measured twice per week using a caliper, and the volume was calculated according to the following formula: volume length (mm) × width (mm) × height (mm)/2. Tumor weight and volume are presented as means ± SD from eight mice. (B) Immunohistochemistry was used to determine the expression levels of proliferating cell nuclear antigen in the melanoma tissues of PA-treated IL-4 mice and AOO-treated IL-4 mice. (C) The expression of IL-4 and apoptotic proteins was detected by Western blotting using specific antibodies: IL-4, IL-4Rα, JAK1, p-JAK1, cleaved caspase-3, -8, -9, BCL-2, and β-actin; β-actin was used as an internal control. (D) STAT6 activity in tumor tissues was detected by EMSA, and (E) the nuclear location of STAT6 was determined by Western blotting; histone protein was used as an internal control. All values represent the mean ± SD from eight animal tumor sections. * ( P

Techniques Used: Inhibition, Mouse Assay, Immunohistochemistry, Expressing, Western Blot, Activity Assay

Effects of IL-4 on LPS- or TNF-α–induced STAT6 activation in human keratinocytes. (A and B) STAT6 activity was detected by EMSA. (C and D) The expression of STAT6 and p-STAT6 was detected by Western blotting using specific antibodies. Histone protein was used as an internal control. Each blot is representative of three experiments.
Figure Legend Snippet: Effects of IL-4 on LPS- or TNF-α–induced STAT6 activation in human keratinocytes. (A and B) STAT6 activity was detected by EMSA. (C and D) The expression of STAT6 and p-STAT6 was detected by Western blotting using specific antibodies. Histone protein was used as an internal control. Each blot is representative of three experiments.

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

Apoptotic effect of IL-4 on LPS- or TNF-α–induced cell viability and apoptotic protein expression in human keratinocytes. IL-4–treated human keratinocytes were treated with LPS (1 μg/ml) or TNF-α (10 ng/ml). After treatment, cell viability was measured by the MTT assay (A and B), and the expression of apoptosis regulatory proteins (C and D) was determined. β-Actin was used as an internal control. Each blot is representative of three experiments. # ( P
Figure Legend Snippet: Apoptotic effect of IL-4 on LPS- or TNF-α–induced cell viability and apoptotic protein expression in human keratinocytes. IL-4–treated human keratinocytes were treated with LPS (1 μg/ml) or TNF-α (10 ng/ml). After treatment, cell viability was measured by the MTT assay (A and B), and the expression of apoptosis regulatory proteins (C and D) was determined. β-Actin was used as an internal control. Each blot is representative of three experiments. # ( P

Techniques Used: Expressing, MTT Assay

PA-induced allergy reaction. Induction of the allergenic response to PA in IL-4 mice and AOO-treated IL-4 control mice. (A) IL-4 levels in the serum of Luc/IL-4/CNS-1 transgenic mice and nontransgenic (Non-Tg) mice. The concentration of IL-4 in the Non-Tg mice was 20.34 pg/mg. Changes in various immune-related factors such as ear thickness (B), IgE concentration (C), IL-4 concentration (D), and increases in epidermal thickness in the ear tissue (E) and skin tissue of the dorsal region (F) were detected. * ( P
Figure Legend Snippet: PA-induced allergy reaction. Induction of the allergenic response to PA in IL-4 mice and AOO-treated IL-4 control mice. (A) IL-4 levels in the serum of Luc/IL-4/CNS-1 transgenic mice and nontransgenic (Non-Tg) mice. The concentration of IL-4 in the Non-Tg mice was 20.34 pg/mg. Changes in various immune-related factors such as ear thickness (B), IgE concentration (C), IL-4 concentration (D), and increases in epidermal thickness in the ear tissue (E) and skin tissue of the dorsal region (F) were detected. * ( P

Techniques Used: Mouse Assay, Transgenic Assay, Concentration Assay

Effect of IL-4–activated NK-92MI cells on the expression of apoptosis regulatory proteins in human melanoma cells and colocalization with CD16 and p-STAT6 in human tissues. The SK-MEL-28 human melanoma cells were co-cultured with NK-92MI cells treated with IL-4 (25 ng/ml). After treatment, (A) cell viability was measured by the MTT assay, and (B) morphological changes were observed. Values are the mean ± SD of three experiments with replicates. *( P
Figure Legend Snippet: Effect of IL-4–activated NK-92MI cells on the expression of apoptosis regulatory proteins in human melanoma cells and colocalization with CD16 and p-STAT6 in human tissues. The SK-MEL-28 human melanoma cells were co-cultured with NK-92MI cells treated with IL-4 (25 ng/ml). After treatment, (A) cell viability was measured by the MTT assay, and (B) morphological changes were observed. Values are the mean ± SD of three experiments with replicates. *( P

Techniques Used: Expressing, Cell Culture, MTT Assay

Reversed effect of IL-4 siRNA on IL-4–induced cell growth inhibition and STAT6 activation. The melanoma cells were transfected with IL-4 siRNA (100 nM) for 24 hours, and the cells were then treated with IL-4 (50 ng/ml) for 24 hours. After treatment, (A) cell viability was measured by the MTT assay, and (B) the expression of apoptosis regulatory proteins and (C) STAT6 activity were determined as described in the legend to Figure 6 ; histone protein was used as an internal control. Cell growth is presented as the means ± SD of three experiments. *( P
Figure Legend Snippet: Reversed effect of IL-4 siRNA on IL-4–induced cell growth inhibition and STAT6 activation. The melanoma cells were transfected with IL-4 siRNA (100 nM) for 24 hours, and the cells were then treated with IL-4 (50 ng/ml) for 24 hours. After treatment, (A) cell viability was measured by the MTT assay, and (B) the expression of apoptosis regulatory proteins and (C) STAT6 activity were determined as described in the legend to Figure 6 ; histone protein was used as an internal control. Cell growth is presented as the means ± SD of three experiments. *( P

Techniques Used: Inhibition, Activation Assay, Transfection, MTT Assay, Expressing, Activity Assay

Effect of allergy responses on the infiltration of immune cells in tumor tissues. (A) Immunohistochemistry was used to determine the expression levels of IL-4, CD4, CD19, and CD16 in the tissues of PA-treated IL-4 mice and AOO-treated IL-4 mice. (B–F) Percentages of IL-4–, CD4-, CD19-, and CD16-positive cells of the total number of cells counted in the AOO group. (F–J) Changes in various cytokine levels, such as IFN-γ (F), IL-4 (G), IL-1β (H), IL-6 (I), and TNF-α (J). The results are presented as the mean ± SEM from eight mice. * ( P
Figure Legend Snippet: Effect of allergy responses on the infiltration of immune cells in tumor tissues. (A) Immunohistochemistry was used to determine the expression levels of IL-4, CD4, CD19, and CD16 in the tissues of PA-treated IL-4 mice and AOO-treated IL-4 mice. (B–F) Percentages of IL-4–, CD4-, CD19-, and CD16-positive cells of the total number of cells counted in the AOO group. (F–J) Changes in various cytokine levels, such as IFN-γ (F), IL-4 (G), IL-1β (H), IL-6 (I), and TNF-α (J). The results are presented as the mean ± SEM from eight mice. * ( P

Techniques Used: Immunohistochemistry, Expressing, Mouse Assay

32) Product Images from "Fingerprints of CD8+ T cells on human pre-plasma and memory B cells"

Article Title: Fingerprints of CD8+ T cells on human pre-plasma and memory B cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0208187

CD8 positive B cells release cytokines and immunoglobulins. (A) Production of the cytokines EBI3 and IL-35 was determined by ELISA from supernatants of freshly sorted CD8+ B cells (black bars) and CD8- control B cells (white bars). Cultures contained 50’000 cells in 200 μl volume. As indicated, samples for both measurements were obtained at 18h and 4 days, without or with CD40+IL-4 stimulation. The dashed line indicates the assay detection limit. (B) Production of IgG was determined by ELISPOT using cells sorted either from apheresis material or from peripheral blood (PBMC). Spots were counted, and are expressed as percentage of input cells (3’000 or 1’000 cells per well, as indicated). Cultures were performed without (black bars) or with CD40L+IL-4 stimulation (white bars).
Figure Legend Snippet: CD8 positive B cells release cytokines and immunoglobulins. (A) Production of the cytokines EBI3 and IL-35 was determined by ELISA from supernatants of freshly sorted CD8+ B cells (black bars) and CD8- control B cells (white bars). Cultures contained 50’000 cells in 200 μl volume. As indicated, samples for both measurements were obtained at 18h and 4 days, without or with CD40+IL-4 stimulation. The dashed line indicates the assay detection limit. (B) Production of IgG was determined by ELISPOT using cells sorted either from apheresis material or from peripheral blood (PBMC). Spots were counted, and are expressed as percentage of input cells (3’000 or 1’000 cells per well, as indicated). Cultures were performed without (black bars) or with CD40L+IL-4 stimulation (white bars).

Techniques Used: Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

33) Product Images from "A novel mechanism for ERK-dependent regulation of IL4 transcription during human Th2-cell differentiation"

Article Title: A novel mechanism for ERK-dependent regulation of IL4 transcription during human Th2-cell differentiation

Journal: Immunology and Cell Biology

doi: 10.1038/icb.2011.87

Association of ERK is necessary for IL4 promoter activity. ( a ) Jurkat cells were transfected with the pEGFP1 vector (vector, top two panels), with the pIL-4-EGFP construct (from second to the fourth row of panels) or with a derivative of the pIL-4-EGFP construct in which the ERK-binding region was partially deleted (Pdel-IL-4-EGFP, bottom panels). This included two additional experimental groups in which pIL-4-EGF-transfected cells were also treated with either ERK-specific siRNA (panels in the third row from top) or with the MEK inhibitor U0126 (panels in the fourth row from top) as indicated. Figure shows the level of fluorescence obtained in each of these groups of cells when they were either left unstimulated or stimulated through the TCR for 24 h. Values indicated in each panel are the mean (±s.e.m) intensity of fluorescence measured for at least 40 cells from different fields in 3 separate slides for each group. ( b ) Naive CD4 + T cells were either left untreated (top panel) or treated with either ERK1/2 siRNA (KD) or U0126 (lower two panels). These cells were then stimulated through the TCR, and time-dependent recruitment of RNA polymerase II (RNAP II) to the IL4 promoter was monitored by ChIP analysis. The corresponding profiles obtained without anti-ERK antibody (no Ab) and input are also shown. ( c ) Time-dependent recruitment of indicated transcription factors, as determined by ChIP, to the IL-4 promoter after stimulation of cells through the TCR either in the presence (+) or absence (−) of U0126.
Figure Legend Snippet: Association of ERK is necessary for IL4 promoter activity. ( a ) Jurkat cells were transfected with the pEGFP1 vector (vector, top two panels), with the pIL-4-EGFP construct (from second to the fourth row of panels) or with a derivative of the pIL-4-EGFP construct in which the ERK-binding region was partially deleted (Pdel-IL-4-EGFP, bottom panels). This included two additional experimental groups in which pIL-4-EGF-transfected cells were also treated with either ERK-specific siRNA (panels in the third row from top) or with the MEK inhibitor U0126 (panels in the fourth row from top) as indicated. Figure shows the level of fluorescence obtained in each of these groups of cells when they were either left unstimulated or stimulated through the TCR for 24 h. Values indicated in each panel are the mean (±s.e.m) intensity of fluorescence measured for at least 40 cells from different fields in 3 separate slides for each group. ( b ) Naive CD4 + T cells were either left untreated (top panel) or treated with either ERK1/2 siRNA (KD) or U0126 (lower two panels). These cells were then stimulated through the TCR, and time-dependent recruitment of RNA polymerase II (RNAP II) to the IL4 promoter was monitored by ChIP analysis. The corresponding profiles obtained without anti-ERK antibody (no Ab) and input are also shown. ( c ) Time-dependent recruitment of indicated transcription factors, as determined by ChIP, to the IL-4 promoter after stimulation of cells through the TCR either in the presence (+) or absence (−) of U0126.

Techniques Used: Activity Assay, Transfection, Plasmid Preparation, Construct, Binding Assay, Fluorescence, Chromatin Immunoprecipitation

The effect of ERK on IL4 gene expression is specific and involves both ERK-1 and ERK-2. ( a ) compares the distribution of three transcription factors (GATA3, NFATc1 and c-Jun) between the cytoplasmic and nuclear fractions in unstimulated (UNST), TCR-triggered (TCR, stimulation time of 30 min) and U0126-treated cells, followed by TCR triggering (U0126+TCR, stimulation time of 30 min). LC denotes the loading control, which was PLCγ2 for cytoplasmic fractions and Histone 1 for the nuclear fractions. ( b ) Effects of treatment of cells with either GFP or ERK1/2-specific siRNA on induction of the IL-13 transcript in activated cells. The time points at which transcript levels were determined by RT-PCR are indicated. Here, primers specific for β-actin were also included to provide an internal normalization control. ( c ) Results from a ChIP assay that monitored the recruitment of NFATc, c-jun and GATA3 to the region spanning −76 to −234 nt of the IL-13 promoter. Here, either untreated cells (U0126−) or cells treated with U0126 (U0126+) were stimulated through the TCR as indicated. Input and no Ab controls are also shown. ( d ) Results of experiments in which cells were activated through the TCR for indicated time and then subjected to a ChIP analysis using antibodies that were either specific only to ERK-1 or to ERK-2. For the purposes of comparison, an additional group was also included in which antibodies to both ERK-1 and ERK-2 were combined in the ChIP experiment (ERK1/2). Results shown are a representative of three separate experiments. ( e ) gives the results of a DNA pull-down experiment in which nuclear extracts from CD4 + T cells, stimulated through the TCR for 3 h, were incubated either with a 20-mer double-stranded DNA probe extracted from Figure 3d or with a control probe of similar length. In both cases, the probes were biotinylated. After incubation, the probes were separated by affinity chromatography (streptavidin-agarose), and the bound proteins eluted and analyzed for the presence of ERK-1 and ERK-2 by western blot analysis (see the ‘Methods' section). Results shown are from one of three separate experiments. ( f ) Results obtained in an RT-PCR experiment in which cells transfected separately with ERK1 siRNA (ERK1), ERK2 siRNA (ERK2) or with a combination of both (ERK1/2) were stimulated through the TCR. At the end of 84 h, cells were harvested, the total RNA was isolated and analyzed by RT-PCR for the IL-4 transcript. For the purposes of comparison, results obtained in cells that were either not treated with any siRNA (Control) or those treated with siRNA specific for GFP (Mock) are also shown. In all cases, primers specific for β-actin were also included to provide an internal normalization control.
Figure Legend Snippet: The effect of ERK on IL4 gene expression is specific and involves both ERK-1 and ERK-2. ( a ) compares the distribution of three transcription factors (GATA3, NFATc1 and c-Jun) between the cytoplasmic and nuclear fractions in unstimulated (UNST), TCR-triggered (TCR, stimulation time of 30 min) and U0126-treated cells, followed by TCR triggering (U0126+TCR, stimulation time of 30 min). LC denotes the loading control, which was PLCγ2 for cytoplasmic fractions and Histone 1 for the nuclear fractions. ( b ) Effects of treatment of cells with either GFP or ERK1/2-specific siRNA on induction of the IL-13 transcript in activated cells. The time points at which transcript levels were determined by RT-PCR are indicated. Here, primers specific for β-actin were also included to provide an internal normalization control. ( c ) Results from a ChIP assay that monitored the recruitment of NFATc, c-jun and GATA3 to the region spanning −76 to −234 nt of the IL-13 promoter. Here, either untreated cells (U0126−) or cells treated with U0126 (U0126+) were stimulated through the TCR as indicated. Input and no Ab controls are also shown. ( d ) Results of experiments in which cells were activated through the TCR for indicated time and then subjected to a ChIP analysis using antibodies that were either specific only to ERK-1 or to ERK-2. For the purposes of comparison, an additional group was also included in which antibodies to both ERK-1 and ERK-2 were combined in the ChIP experiment (ERK1/2). Results shown are a representative of three separate experiments. ( e ) gives the results of a DNA pull-down experiment in which nuclear extracts from CD4 + T cells, stimulated through the TCR for 3 h, were incubated either with a 20-mer double-stranded DNA probe extracted from Figure 3d or with a control probe of similar length. In both cases, the probes were biotinylated. After incubation, the probes were separated by affinity chromatography (streptavidin-agarose), and the bound proteins eluted and analyzed for the presence of ERK-1 and ERK-2 by western blot analysis (see the ‘Methods' section). Results shown are from one of three separate experiments. ( f ) Results obtained in an RT-PCR experiment in which cells transfected separately with ERK1 siRNA (ERK1), ERK2 siRNA (ERK2) or with a combination of both (ERK1/2) were stimulated through the TCR. At the end of 84 h, cells were harvested, the total RNA was isolated and analyzed by RT-PCR for the IL-4 transcript. For the purposes of comparison, results obtained in cells that were either not treated with any siRNA (Control) or those treated with siRNA specific for GFP (Mock) are also shown. In all cases, primers specific for β-actin were also included to provide an internal normalization control.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Chromatin Immunoprecipitation, Incubation, Affinity Chromatography, Western Blot, Transfection, Isolation

ERK regulates IL4 gene transcription. ( a ) Kinetics of IL-4 mRNA induction in naive CD4 + T cells stimulated with TCR in the presence (TCR+IL-4) or absence (TCR) of IL-4. ( b ) Effects of treatment of cells with either GFP or ERK1/2-specific siRNA on induction of the IL-4 transcript in activated and Th2-polarizing cells. The time points at which transcript levels were determined by RT-PCR are indicated. Here, primers specific for β-actin were also included to provide an internal normalization control. Western blots in c confirm the silencing of STAT6 protein levels in the nuclear extract of cells treated for the indicated times with specific siRNA. Blots were also probed with anti-histone H3 antibodies to provide a loading control. The effects of STAT6- versus GFP-silencing on IL-4 mRNA levels as a function of time are shown in d . In e , naive CD4 + T cells were stimulated under Th2-polarizing conditions either in the absence (−) or presence (+) of U0126, and IL-4 transcript levels determined by RT-PCR 84 h later.
Figure Legend Snippet: ERK regulates IL4 gene transcription. ( a ) Kinetics of IL-4 mRNA induction in naive CD4 + T cells stimulated with TCR in the presence (TCR+IL-4) or absence (TCR) of IL-4. ( b ) Effects of treatment of cells with either GFP or ERK1/2-specific siRNA on induction of the IL-4 transcript in activated and Th2-polarizing cells. The time points at which transcript levels were determined by RT-PCR are indicated. Here, primers specific for β-actin were also included to provide an internal normalization control. Western blots in c confirm the silencing of STAT6 protein levels in the nuclear extract of cells treated for the indicated times with specific siRNA. Blots were also probed with anti-histone H3 antibodies to provide a loading control. The effects of STAT6- versus GFP-silencing on IL-4 mRNA levels as a function of time are shown in d . In e , naive CD4 + T cells were stimulated under Th2-polarizing conditions either in the absence (−) or presence (+) of U0126, and IL-4 transcript levels determined by RT-PCR 84 h later.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Western Blot

ERK associates with the IL4 promoter in activated T cells. ( a ) IL-4 spot-forming cells (SFCs) from an ELISPOT assay in which naive CD4 + T cells were either transfected with GFP siRNA (Group 1), ERK siRNA (Group 2) or ERK siRNA, followed by supplementation of recombinant IL-4 (Group 3) in the culture. ‘Un' represents unstimulated cells, whereas TCR and TCR+IL-4 represent activation and polarizing conditions, respectively. * P ⩽0.0001. Each bar represents the data from three independent experiments (mean±s.d. value), performed on three biological replicates in which each replicate represented a pool of cells isolated from 3–5 individuals. ( b ) Naive CD4 + T cells were either left untreated (top panel) or treated with either ERK1/2 siRNA or U0126 (lower two panels). These cells were then stimulated through the TCR, and time-dependent recruitment of ERK to the IL4 promoter was monitored by ChIP analysis. The corresponding profiles obtained without anti-ERK antibody (no Ab) and input are also shown. ( c ) ERK-specific chromatin-immunoprecipitated samples from either TCR-activated (3 h) or unstimulated cells were hybridized against a tiled array that was generated around the 60-nucleotide long IL-4 promoter region from −1374 to −1315 nt. This region included the ERK-binding site. The first probe in this array was a 60 nt sequence spanning from nt positions −1404 to −1345. All subsequent probes—also 60 nt long—then represented successive one-nucleotide shifts in sequence, terminating with a probe extending from −1344 to −1285 nt. The fold enhancement in hybridization intensity—in terms of log 2 values—obtained for each of these probes with sample from TCR-activated cells, relative to that obtained with a parallel sample from unstimulated cells, is plotted here. The individual probes are identified on the X axis on the basis of the sequence position of the central nucleotide. ( d ) Parent 60 nt region where the optimal ERK-binding domain is highlighted in red.
Figure Legend Snippet: ERK associates with the IL4 promoter in activated T cells. ( a ) IL-4 spot-forming cells (SFCs) from an ELISPOT assay in which naive CD4 + T cells were either transfected with GFP siRNA (Group 1), ERK siRNA (Group 2) or ERK siRNA, followed by supplementation of recombinant IL-4 (Group 3) in the culture. ‘Un' represents unstimulated cells, whereas TCR and TCR+IL-4 represent activation and polarizing conditions, respectively. * P ⩽0.0001. Each bar represents the data from three independent experiments (mean±s.d. value), performed on three biological replicates in which each replicate represented a pool of cells isolated from 3–5 individuals. ( b ) Naive CD4 + T cells were either left untreated (top panel) or treated with either ERK1/2 siRNA or U0126 (lower two panels). These cells were then stimulated through the TCR, and time-dependent recruitment of ERK to the IL4 promoter was monitored by ChIP analysis. The corresponding profiles obtained without anti-ERK antibody (no Ab) and input are also shown. ( c ) ERK-specific chromatin-immunoprecipitated samples from either TCR-activated (3 h) or unstimulated cells were hybridized against a tiled array that was generated around the 60-nucleotide long IL-4 promoter region from −1374 to −1315 nt. This region included the ERK-binding site. The first probe in this array was a 60 nt sequence spanning from nt positions −1404 to −1345. All subsequent probes—also 60 nt long—then represented successive one-nucleotide shifts in sequence, terminating with a probe extending from −1344 to −1285 nt. The fold enhancement in hybridization intensity—in terms of log 2 values—obtained for each of these probes with sample from TCR-activated cells, relative to that obtained with a parallel sample from unstimulated cells, is plotted here. The individual probes are identified on the X axis on the basis of the sequence position of the central nucleotide. ( d ) Parent 60 nt region where the optimal ERK-binding domain is highlighted in red.

Techniques Used: Enzyme-linked Immunospot, Transfection, Recombinant, Activation Assay, Isolation, Chromatin Immunoprecipitation, Immunoprecipitation, Generated, Binding Assay, Sequencing, Hybridization

ERK selectively regulates Th2-cell differentiation. ( a ) Results of a western blot analysis, at indicated times, for ERK-1/2 in naive CD4 + T cells transfected with ERK-specific siRNA. The effect of treatment of GFP-specific siRNA on ERK levels, at 36 h later, is also included, with GAPDH as an internal loading control. ( b , c ) Effect of treatment of naive CD4 + T cells with siRNA specific for either ERK or GFP on the generation of either IL-4 and IL-13 (panel b ) or IFN-γ (panel c ) cytokine-producing cells as measured by an ELISPOT assay at day 7 of culture. The stimulation conditions tested here were activation (TCR), Th2 polarization (TCR+IL-4) and Th1 polarization (TCR+IL-12). The corresponding profiles obtained in mock (GFP)-siRNA-treated cells cultured under similar conditions are also shown. * P ⩽0.05 (one-way analysis of variance (ANOVA) test); NS=not significant. Each bar represents the mean (±s.d.) of three biological replicates in which each replicate represented a pool of cells isolated from 3 to 5 individuals. In a separate experiment, we stimulated either mock- (red line) or ERK-silenced (blue line) cells under Th2-polarizing conditions for 7 days and then measured the extent of apoptotic cell death by staining with Annexin-V, followed by flow-cytometric analysis. The profiles obtained are shown in d and represents one of three separate experiments. For e , a similar experiment was performed, except that cells were first labeled with CFSE before stimulation and the extent of cell proliferation was determined 7 days later ( n =3). f compares the concentration of IL-2 present in the supernatant of these cultures at day 7. Here, U indicates cells that were not activated, whereas S denotes the stimulated groups. Values are the mean (±s.d.) of three separate experiments.
Figure Legend Snippet: ERK selectively regulates Th2-cell differentiation. ( a ) Results of a western blot analysis, at indicated times, for ERK-1/2 in naive CD4 + T cells transfected with ERK-specific siRNA. The effect of treatment of GFP-specific siRNA on ERK levels, at 36 h later, is also included, with GAPDH as an internal loading control. ( b , c ) Effect of treatment of naive CD4 + T cells with siRNA specific for either ERK or GFP on the generation of either IL-4 and IL-13 (panel b ) or IFN-γ (panel c ) cytokine-producing cells as measured by an ELISPOT assay at day 7 of culture. The stimulation conditions tested here were activation (TCR), Th2 polarization (TCR+IL-4) and Th1 polarization (TCR+IL-12). The corresponding profiles obtained in mock (GFP)-siRNA-treated cells cultured under similar conditions are also shown. * P ⩽0.05 (one-way analysis of variance (ANOVA) test); NS=not significant. Each bar represents the mean (±s.d.) of three biological replicates in which each replicate represented a pool of cells isolated from 3 to 5 individuals. In a separate experiment, we stimulated either mock- (red line) or ERK-silenced (blue line) cells under Th2-polarizing conditions for 7 days and then measured the extent of apoptotic cell death by staining with Annexin-V, followed by flow-cytometric analysis. The profiles obtained are shown in d and represents one of three separate experiments. For e , a similar experiment was performed, except that cells were first labeled with CFSE before stimulation and the extent of cell proliferation was determined 7 days later ( n =3). f compares the concentration of IL-2 present in the supernatant of these cultures at day 7. Here, U indicates cells that were not activated, whereas S denotes the stimulated groups. Values are the mean (±s.d.) of three separate experiments.

Techniques Used: Cell Differentiation, Western Blot, Transfection, Enzyme-linked Immunospot, Activation Assay, Cell Culture, Isolation, Staining, Flow Cytometry, Labeling, Concentration Assay

Stochastic variability in ERK levels influences individual-specific variations in susceptibility to Th2-cell differentiation. ( a ) Extent of variability in ERK1/2 protein levels in unstimulated naive CD4 + T cells. ERK1/2 levels were determined from western blot analysis using antibodies specific for both ERK-1 and ERK-2. Here, each bar depicts the results for one individual ( n =38). For the purposes of comparison, GAPDH levels were also monitored in each of these samples and the panel shows the comparison of the relative variability of total ERK1/2 levels, in comparison with that of GAPDH. The Y axis gives the normalized intensity of the protein band in each sample, after subtracting the mean value of the band intensities in all the samples (X−X mean ). The corresponding values for variance in X−X mean for ERK (V ERK ) and GAPDH (V GAPDH ) are indicated. ( b ) Results of a regression analysis between p-ERK/ERK (that is, ratio of phospho-ERK1/2 to ERK1/2 protein) and the frequency of IL-4-producing cells in naive CD4 + T cells obtained from different individuals ( n =20) as described in the text. Here, the frequency of IL-4-producing cells was determined by an ELISPOT assay. Each value represents the mean of triplicate sets.
Figure Legend Snippet: Stochastic variability in ERK levels influences individual-specific variations in susceptibility to Th2-cell differentiation. ( a ) Extent of variability in ERK1/2 protein levels in unstimulated naive CD4 + T cells. ERK1/2 levels were determined from western blot analysis using antibodies specific for both ERK-1 and ERK-2. Here, each bar depicts the results for one individual ( n =38). For the purposes of comparison, GAPDH levels were also monitored in each of these samples and the panel shows the comparison of the relative variability of total ERK1/2 levels, in comparison with that of GAPDH. The Y axis gives the normalized intensity of the protein band in each sample, after subtracting the mean value of the band intensities in all the samples (X−X mean ). The corresponding values for variance in X−X mean for ERK (V ERK ) and GAPDH (V GAPDH ) are indicated. ( b ) Results of a regression analysis between p-ERK/ERK (that is, ratio of phospho-ERK1/2 to ERK1/2 protein) and the frequency of IL-4-producing cells in naive CD4 + T cells obtained from different individuals ( n =20) as described in the text. Here, the frequency of IL-4-producing cells was determined by an ELISPOT assay. Each value represents the mean of triplicate sets.

Techniques Used: Cell Differentiation, Western Blot, Enzyme-linked Immunospot

34) Product Images from "Interaction affinity between cytokine receptor components on the cell surface"

Article Title: Interaction affinity between cytokine receptor components on the cell surface

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

Overlaid dose-response curves for the IL-4-dependent proliferation of PHA-activated T cells measured at various fixed concentrations of ( A ) blocking anti-IL-4Rα mAb at 0 (○) 0.41 (□), 1.23 (▵), 3.7 (◊), 11.1 (▿), 33.3 (⊠), or 100 μg/ml (⊙); ( B ) anti-γ c mAb CP.B8 at 0, (○), 1.23 (□), 3.7 (▵), 11.1 (◊), 33.3 (▿), or 100 μg/ml (⊠); ( C ) isotype controls MOPC 21 (mouse IgG1, for CP.B8) (□) and UPC10 (mouse IgG2a, for anti-IL-4Rα) (▵) at 100 μg/ml, or no mAb (○); or ( D ) anti-γ c mAb CP.B8 at 0, (○), 100 (□), or 300 μg/ml (▵), MOPC 21 at 300 μg/ml (▿), or a CP.B8 Fab fragment at 100 μg/ml (◊). Data are represented as mean counts/min (cpm) determined from triplicate wells. The solid lines are best fits of the data to a standard four-parameter equation. ( Inset A ) EC 50 for IL-4 increases linearly with anti-IL-4Rα mAb concentration. ( Inset B ) EC 50 for IL-4 is independent of CP.B8 concentration.
Figure Legend Snippet: Overlaid dose-response curves for the IL-4-dependent proliferation of PHA-activated T cells measured at various fixed concentrations of ( A ) blocking anti-IL-4Rα mAb at 0 (○) 0.41 (□), 1.23 (▵), 3.7 (◊), 11.1 (▿), 33.3 (⊠), or 100 μg/ml (⊙); ( B ) anti-γ c mAb CP.B8 at 0, (○), 1.23 (□), 3.7 (▵), 11.1 (◊), 33.3 (▿), or 100 μg/ml (⊠); ( C ) isotype controls MOPC 21 (mouse IgG1, for CP.B8) (□) and UPC10 (mouse IgG2a, for anti-IL-4Rα) (▵) at 100 μg/ml, or no mAb (○); or ( D ) anti-γ c mAb CP.B8 at 0, (○), 100 (□), or 300 μg/ml (▵), MOPC 21 at 300 μg/ml (▿), or a CP.B8 Fab fragment at 100 μg/ml (◊). Data are represented as mean counts/min (cpm) determined from triplicate wells. The solid lines are best fits of the data to a standard four-parameter equation. ( Inset A ) EC 50 for IL-4 increases linearly with anti-IL-4Rα mAb concentration. ( Inset B ) EC 50 for IL-4 is independent of CP.B8 concentration.

Techniques Used: Blocking Assay, Concentration Assay

Binding of 125 I-labeled IL-4 ( A ) to Cos-7 cells transiently transfected with a pCDM8 vector containing the cDNA sequence for full-length huIL-4Rα (2 μg, ○), full-length human γ c (20 μg, ◊), cotransfected with IL-4Rα (2 μg) plus γ c (20 μg, □), or to mock-transfected cells (▵); or ( B ) to PHA-activated (○) or resting (•) PBMC. The data are plotted as the average of duplicate measurements after correction for nonspecific binding. Data are fitted to a simple hyperbolic equation representing a single site binding model. ( Inset A ) Data for IL-4 binding to cells transfected with IL-4Rα alone (○), or cotransfected with IL-4Rα plus γ c (□), represented as Scatchard plots of bound/free 125 I-IL-4 × 10 −4 (B/F) versus bound 125 I-IL-4 (B, in cpm).
Figure Legend Snippet: Binding of 125 I-labeled IL-4 ( A ) to Cos-7 cells transiently transfected with a pCDM8 vector containing the cDNA sequence for full-length huIL-4Rα (2 μg, ○), full-length human γ c (20 μg, ◊), cotransfected with IL-4Rα (2 μg) plus γ c (20 μg, □), or to mock-transfected cells (▵); or ( B ) to PHA-activated (○) or resting (•) PBMC. The data are plotted as the average of duplicate measurements after correction for nonspecific binding. Data are fitted to a simple hyperbolic equation representing a single site binding model. ( Inset A ) Data for IL-4 binding to cells transfected with IL-4Rα alone (○), or cotransfected with IL-4Rα plus γ c (□), represented as Scatchard plots of bound/free 125 I-IL-4 × 10 −4 (B/F) versus bound 125 I-IL-4 (B, in cpm).

Techniques Used: Binding Assay, Labeling, Transfection, Plasmid Preparation, Sequencing

Binding of 125 I-labeled IL-4 to PHA-activated PBMC in the presence of various fixed concentrations of ( A ) blocking anti-IL-4Rα mAb at 0 (○) 0.01 (□), 0.1 (▵), 1.0 (◊), 10 (▿), 100 (⊠), or 1,000 ng/ml (⊙); ( B ) anti-γ c mAb CP.B8 at 0, (○), 0.01 (□), 0.1 (▵), 1.0 (◊), 10 (▿), or 100 μg/ml (⊠); or ( C ) isotype controls MOPC 21 (for CP.B8, □) and UPC10 (for anti-IL-4Rα, ▵) at 100 μg/ml, or no mAb (○). Data are fitted to a simple hyperbolic binding curve.
Figure Legend Snippet: Binding of 125 I-labeled IL-4 to PHA-activated PBMC in the presence of various fixed concentrations of ( A ) blocking anti-IL-4Rα mAb at 0 (○) 0.01 (□), 0.1 (▵), 1.0 (◊), 10 (▿), 100 (⊠), or 1,000 ng/ml (⊙); ( B ) anti-γ c mAb CP.B8 at 0, (○), 0.01 (□), 0.1 (▵), 1.0 (◊), 10 (▿), or 100 μg/ml (⊠); or ( C ) isotype controls MOPC 21 (for CP.B8, □) and UPC10 (for anti-IL-4Rα, ▵) at 100 μg/ml, or no mAb (○). Data are fitted to a simple hyperbolic binding curve.

Techniques Used: Binding Assay, Labeling, Blocking Assay

). In the case of the IL-4 receptor, R 1 is the IL-4Rα chain and R 2 is γ c . For homodimeric receptors such as hGH-R, R 1 and R 2 are identical.
Figure Legend Snippet: ). In the case of the IL-4 receptor, R 1 is the IL-4Rα chain and R 2 is γ c . For homodimeric receptors such as hGH-R, R 1 and R 2 are identical.

Techniques Used:

35) Product Images from "T-Cell Protein Tyrosine Phosphatase, Distinctively Expressed in Activated-B-Cell-Like Diffuse Large B-Cell Lymphomas, Is the Nuclear Phosphatase of STAT6 ▿"

Article Title: T-Cell Protein Tyrosine Phosphatase, Distinctively Expressed in Activated-B-Cell-Like Diffuse Large B-Cell Lymphomas, Is the Nuclear Phosphatase of STAT6 ▿

Journal:

doi: 10.1128/MCB.01234-06

TCPTP-45 inhibits IL-4-induced gene transcription. (A) STAT6-driven luciferase reporter construct C/EBP-N4, STAT6, pRL, and either the EGFP-TCPTP-45 or EGFP plasmid were coexpressed in the HEK 293 cells. For each transfection, 4 μg of each expression
Figure Legend Snippet: TCPTP-45 inhibits IL-4-induced gene transcription. (A) STAT6-driven luciferase reporter construct C/EBP-N4, STAT6, pRL, and either the EGFP-TCPTP-45 or EGFP plasmid were coexpressed in the HEK 293 cells. For each transfection, 4 μg of each expression

Techniques Used: Luciferase, Construct, Plasmid Preparation, Transfection, Expressing

IL-4 induces enhanced STAT6 phosphorylation in TCPTP-deficient cells. (A) Mouse embryo fibroblasts from TCPTP-deficient (−/−), wild-type (+/+), and heterozygous (−/+) mice were serum starved for 4 h and
Figure Legend Snippet: IL-4 induces enhanced STAT6 phosphorylation in TCPTP-deficient cells. (A) Mouse embryo fibroblasts from TCPTP-deficient (−/−), wild-type (+/+), and heterozygous (−/+) mice were serum starved for 4 h and

Techniques Used: Mouse Assay

Analysis of dephosphorylation of STAT6 in GCB-like and ABC-like DLBCL cells. (A) GCB-like (SUDHL6) and ABC-like (OCILY10) DLBCL cell lines were treated with IL-4 (100 U/ml) for 15 min, followed by a staurosporine chase for various time periods (in minutes)
Figure Legend Snippet: Analysis of dephosphorylation of STAT6 in GCB-like and ABC-like DLBCL cells. (A) GCB-like (SUDHL6) and ABC-like (OCILY10) DLBCL cell lines were treated with IL-4 (100 U/ml) for 15 min, followed by a staurosporine chase for various time periods (in minutes)

Techniques Used: De-Phosphorylation Assay

TCPTP-45 interacts with STAT6 via its catalytic domain. (A) STAT6 and TCPTP(1-349) or GFP-tagged TCPTP-45 were transiently expressed in the HEK 293 cells. Forty-eight hours posttransfection, the cells were stimulated with IL-4 (100 U/ml) for 30 min. Nuclear
Figure Legend Snippet: TCPTP-45 interacts with STAT6 via its catalytic domain. (A) STAT6 and TCPTP(1-349) or GFP-tagged TCPTP-45 were transiently expressed in the HEK 293 cells. Forty-eight hours posttransfection, the cells were stimulated with IL-4 (100 U/ml) for 30 min. Nuclear

Techniques Used:

IL-4 stimulation increases TCPTP protein levels. (A) VAL DLBCL cells were stimulated with IL-4 (100 U/ml) (+) for 3, 6, and 12 h. Cell lysates were prepared, and TCPTP and actin were immunoblotted. Both the blots and results of densitometry analysis
Figure Legend Snippet: IL-4 stimulation increases TCPTP protein levels. (A) VAL DLBCL cells were stimulated with IL-4 (100 U/ml) (+) for 3, 6, and 12 h. Cell lysates were prepared, and TCPTP and actin were immunoblotted. Both the blots and results of densitometry analysis

Techniques Used:

TCPTP-45 dephosphorylates IL-4-induced STAT6 in the nucleus. (A) HEK 293 cells were transfected (+) with 45-kDa TCPTP plasmid (TCPTP-45 WT) and either STAT6 plasmid or empty vector (Mock Vector). Forty-eight hours after transfection, cells were
Figure Legend Snippet: TCPTP-45 dephosphorylates IL-4-induced STAT6 in the nucleus. (A) HEK 293 cells were transfected (+) with 45-kDa TCPTP plasmid (TCPTP-45 WT) and either STAT6 plasmid or empty vector (Mock Vector). Forty-eight hours after transfection, cells were

Techniques Used: Transfection, Plasmid Preparation

The nucleus-restricted GFP-tagged TCPTP-45 fusion protein dephosphorylates IL-4-induced STAT6 in the nucleus. (A) HEK 293 cells were transfected with pEGFP-TCPTP-C1 plasmid as described in Materials and Methods. Forty eight hours posttransfection, the
Figure Legend Snippet: The nucleus-restricted GFP-tagged TCPTP-45 fusion protein dephosphorylates IL-4-induced STAT6 in the nucleus. (A) HEK 293 cells were transfected with pEGFP-TCPTP-C1 plasmid as described in Materials and Methods. Forty eight hours posttransfection, the

Techniques Used: Transfection, Plasmid Preparation

36) Product Images from "Linking the effects of helminth infection, diet and the gut microbiota with human whole-blood signatures"

Article Title: Linking the effects of helminth infection, diet and the gut microbiota with human whole-blood signatures

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1008066

Helminth dependent covariates of microbial and blood profiles. (A) Blood covariates that responded significantly to deworming, with changes in values that resembled values seen in urban participants, as demonstrated using the multi-group boxplots (left). Values for boxplots are based on 18 urban participants and 46 Orang Asli participants with pre-deworming serum chemistry values, as well as 34 Orang Asli who received deworming treatment and had serum iron and serum globulin levels measured post-deworming and 33 Orang Asli who received deworming treatment and had serum zinc levels measured post-deworming. (Right) Connected line plots for paired comparisons of 30 pairs of pre- and post-deworming samples for serum zinc levels, as well as 31 pairs of pre- and post-deworming samples for serum iron and globulin levels. P-values for two-group comparisons (pre-deworming Orang Asli vs. urban participants) are based on t-tests (when data is normally distributed) and Mann-Whitney tests (when data is not normally distributed). Normality of data distribution is determined using the Shapiro-Wilk test of normality. Statistical significance of deworming changes are based on paired t-test of pre- and post-deworming paired values. Microbial OTUs associated with changes in (B) serum zinc levels and (C) serum iron levels post-deworming treatment are shown; values are coefficients from SPLS-regression models. Selected microbial OTUs are labeled with taxonomic assignments of the finest possible resolution and color coded by taxonomic order. Repeated taxa names represent two distinct OTUs with the same taxonomic assignment. (D) A PCA plot based on the 1835 most variable genes (85 th percentile) shows separation between transcriptional profiles of macrophages that were treated (teal) or untreated (pink) with zinc sulfate. Some samples were treated with IL-4 (squares) and IL-13 (triangles) as well. (E) Heatmap of the 41 immune-related genes (GO:0002376) that are significantly differentially expressed in zinc-stimulated versus unstimulated experiments (cutoff p = 0.01, |log2 fold-change| > 1).
Figure Legend Snippet: Helminth dependent covariates of microbial and blood profiles. (A) Blood covariates that responded significantly to deworming, with changes in values that resembled values seen in urban participants, as demonstrated using the multi-group boxplots (left). Values for boxplots are based on 18 urban participants and 46 Orang Asli participants with pre-deworming serum chemistry values, as well as 34 Orang Asli who received deworming treatment and had serum iron and serum globulin levels measured post-deworming and 33 Orang Asli who received deworming treatment and had serum zinc levels measured post-deworming. (Right) Connected line plots for paired comparisons of 30 pairs of pre- and post-deworming samples for serum zinc levels, as well as 31 pairs of pre- and post-deworming samples for serum iron and globulin levels. P-values for two-group comparisons (pre-deworming Orang Asli vs. urban participants) are based on t-tests (when data is normally distributed) and Mann-Whitney tests (when data is not normally distributed). Normality of data distribution is determined using the Shapiro-Wilk test of normality. Statistical significance of deworming changes are based on paired t-test of pre- and post-deworming paired values. Microbial OTUs associated with changes in (B) serum zinc levels and (C) serum iron levels post-deworming treatment are shown; values are coefficients from SPLS-regression models. Selected microbial OTUs are labeled with taxonomic assignments of the finest possible resolution and color coded by taxonomic order. Repeated taxa names represent two distinct OTUs with the same taxonomic assignment. (D) A PCA plot based on the 1835 most variable genes (85 th percentile) shows separation between transcriptional profiles of macrophages that were treated (teal) or untreated (pink) with zinc sulfate. Some samples were treated with IL-4 (squares) and IL-13 (triangles) as well. (E) Heatmap of the 41 immune-related genes (GO:0002376) that are significantly differentially expressed in zinc-stimulated versus unstimulated experiments (cutoff p = 0.01, |log2 fold-change| > 1).

Techniques Used: MANN-WHITNEY, Labeling

37) Product Images from "Global gene regulation during activation of immunoglobulin class switching in human B cells"

Article Title: Global gene regulation during activation of immunoglobulin class switching in human B cells

Journal: Scientific Reports

doi: 10.1038/srep37988

Expression of five key CSR genes over the 288 hour time course following IL-4 and anti-CD40 stimulation. Abundance is displayed on a log2 scale. Results of triplicates are shown. Time point is in hours. Abbreviations: Transcript Cluster (TC).
Figure Legend Snippet: Expression of five key CSR genes over the 288 hour time course following IL-4 and anti-CD40 stimulation. Abundance is displayed on a log2 scale. Results of triplicates are shown. Time point is in hours. Abbreviations: Transcript Cluster (TC).

Techniques Used: Expressing

38) Product Images from "Alternative splice variants of AID are not stoichiometrically present at the protein level in chronic lymphocytic leukemia"

Article Title: Alternative splice variants of AID are not stoichiometrically present at the protein level in chronic lymphocytic leukemia

Journal: European Journal of Immunology

doi: 10.1002/eji.201343853

AID splicing in primary mouse B cells and mouse CLL cells. Primary mouse B cells were stimulated with (A) LPS/IL-4 or (B) as indicated and retrovirally transduced with the indicated pMx-based vectors. (A) RT-PCR was performed to amplify endogenous AID (AIDendo) or retrovirally expressed AID-SRC (AIDtrans). As control, RT-PCR of endogenous GAPDH was carried out. In a separate experiment, whole cell lysates of transduced mouse cells were subjected to immunoblotting (IB, lower panel). Transgenic AID was detected using GFP-specific antibodies. Detection of tubulin was used as a loading control. Data are representative of two independent experiments. (B) Immunoblot on lysates from WT mouse B cells were stimulated as indicated and retrovirally transduced with pMxAID-SRC using GFP-specific antibodies. AID-FL bands serve as loading controls. HEK293FT n.t. lane represents negative control to detect nonspecific binding of GFP antibodies. Data are from one experiment.
Figure Legend Snippet: AID splicing in primary mouse B cells and mouse CLL cells. Primary mouse B cells were stimulated with (A) LPS/IL-4 or (B) as indicated and retrovirally transduced with the indicated pMx-based vectors. (A) RT-PCR was performed to amplify endogenous AID (AIDendo) or retrovirally expressed AID-SRC (AIDtrans). As control, RT-PCR of endogenous GAPDH was carried out. In a separate experiment, whole cell lysates of transduced mouse cells were subjected to immunoblotting (IB, lower panel). Transgenic AID was detected using GFP-specific antibodies. Detection of tubulin was used as a loading control. Data are representative of two independent experiments. (B) Immunoblot on lysates from WT mouse B cells were stimulated as indicated and retrovirally transduced with pMxAID-SRC using GFP-specific antibodies. AID-FL bands serve as loading controls. HEK293FT n.t. lane represents negative control to detect nonspecific binding of GFP antibodies. Data are from one experiment.

Techniques Used: Transduction, Reverse Transcription Polymerase Chain Reaction, Transgenic Assay, Negative Control, Binding Assay

Alternative AID splicing in primary CLL cells and MEC1 cells. RNA and protein were extracted from primary CLL samples that were stimulated with IL-2, IL-4, and CpG (+) or left unstimulated (–) for 6 days. Results of PCR on cDNA using AID-specific and GAPDH-specific primers as loading control are shown (upper panel). Schematic presentations of the alternative splice variants are indicated right to each PCR band. PCR on TOPO-cloned splice variants as templates was performed as size controls and loaded in the first four lanes of the gel. The lower panel shows an immunoblot (IB) on cell lysates using AID- and tubulin-specific antibodies as loading control. Data are representative of two independent experiments.
Figure Legend Snippet: Alternative AID splicing in primary CLL cells and MEC1 cells. RNA and protein were extracted from primary CLL samples that were stimulated with IL-2, IL-4, and CpG (+) or left unstimulated (–) for 6 days. Results of PCR on cDNA using AID-specific and GAPDH-specific primers as loading control are shown (upper panel). Schematic presentations of the alternative splice variants are indicated right to each PCR band. PCR on TOPO-cloned splice variants as templates was performed as size controls and loaded in the first four lanes of the gel. The lower panel shows an immunoblot (IB) on cell lysates using AID- and tubulin-specific antibodies as loading control. Data are representative of two independent experiments.

Techniques Used: Polymerase Chain Reaction, Clone Assay

39) Product Images from "A Novel in vitro Human Macrophage Model to Study the Persistence of Mycobacterium tuberculosis Using Vitamin D3 and Retinoic Acid Activated THP-1 Macrophages"

Article Title: A Novel in vitro Human Macrophage Model to Study the Persistence of Mycobacterium tuberculosis Using Vitamin D3 and Retinoic Acid Activated THP-1 Macrophages

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2011.00067

Activation with RAVD but not cell fusion-inducing cytokines leads to long lived MNGCs containing M. tuberculosis . (A1) Survival of Mtb infected THPs measured after treatment with either recombinant human IL-4 or GM-CSF or their combination with RAVD. Fluorescent conversion of the vital dye alamar blue was used to monitor viability of THPs (100% viability = 100 AFUs from 24-well cultures measured at 485/530 nm using Ascent fluoroscan). Only THPs treated with RAVD-THPs prolong survival measured up to 40 days in this experiment. Those treated with IL-4 or GM-CSF die because of excess growth of Mtb (not shown). (A2) RAVD cells of (A1) examined under phase contrast shows a MNGC (arrow) fusing with single THPs (arrowhead) on day 45. (A3,4) Preformed (day 45) adherent MNGCs (pale green; white arrows) that contain multiple gfp H37Rv fuse with freshly added uninfected, carboxyfluorescein- diacetate (CFSE) stained THPs (T; arrowheads) to form expanded MNGCs (bar = 5 μM). (A5) Phase contrast images of MNGCs on day 60 tend to show syncytium formation (bars = 5 μM). (B) Supernatants of RAVD MNGCs of (A1) collected on days of culture shown were measured for cytokine–chemokines using sandwich ELISA. RAVD activated and Mtb infected THPs show an initial burst of cytokines and later burst of chemokines.
Figure Legend Snippet: Activation with RAVD but not cell fusion-inducing cytokines leads to long lived MNGCs containing M. tuberculosis . (A1) Survival of Mtb infected THPs measured after treatment with either recombinant human IL-4 or GM-CSF or their combination with RAVD. Fluorescent conversion of the vital dye alamar blue was used to monitor viability of THPs (100% viability = 100 AFUs from 24-well cultures measured at 485/530 nm using Ascent fluoroscan). Only THPs treated with RAVD-THPs prolong survival measured up to 40 days in this experiment. Those treated with IL-4 or GM-CSF die because of excess growth of Mtb (not shown). (A2) RAVD cells of (A1) examined under phase contrast shows a MNGC (arrow) fusing with single THPs (arrowhead) on day 45. (A3,4) Preformed (day 45) adherent MNGCs (pale green; white arrows) that contain multiple gfp H37Rv fuse with freshly added uninfected, carboxyfluorescein- diacetate (CFSE) stained THPs (T; arrowheads) to form expanded MNGCs (bar = 5 μM). (A5) Phase contrast images of MNGCs on day 60 tend to show syncytium formation (bars = 5 μM). (B) Supernatants of RAVD MNGCs of (A1) collected on days of culture shown were measured for cytokine–chemokines using sandwich ELISA. RAVD activated and Mtb infected THPs show an initial burst of cytokines and later burst of chemokines.

Techniques Used: Activation Assay, Infection, Recombinant, Staining, Sandwich ELISA

40) Product Images from "Predominant type-2 response in infants with respiratory syncytial virus (RSV) infection demonstrated by cytokine flow cytometry"

Article Title: Predominant type-2 response in infants with respiratory syncytial virus (RSV) infection demonstrated by cytokine flow cytometry

Journal: Clinical and Experimental Immunology

doi: 10.1046/j.1365-2249.2000.01297.x

Recombinant IL-4 blocks the binding of anti-IL-4 antibody with intracellular IL-4. Intracellular IL-4 staining following polyclonal stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin (thick line), and after the blocking of anti-IL-4 antibody by recombinant human IL-4 (thin line).
Figure Legend Snippet: Recombinant IL-4 blocks the binding of anti-IL-4 antibody with intracellular IL-4. Intracellular IL-4 staining following polyclonal stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin (thick line), and after the blocking of anti-IL-4 antibody by recombinant human IL-4 (thin line).

Techniques Used: Recombinant, Binding Assay, Staining, Blocking Assay

The percentage of CD4 lymphocytes producing IL-4 (a) and IFN-γ (c), and percentage of CD8 lymphocytes producing IL-4 (b) and IFN-γ (d) upon polyclonal stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin after a 6-h incubation in controls ( n = 10) and RSV-infected infants ( n = 30). Each symbol characterizes one individual tested. The lines represent medians. The non-parametric Mann–Whitney U -test was used for between-group comparison.
Figure Legend Snippet: The percentage of CD4 lymphocytes producing IL-4 (a) and IFN-γ (c), and percentage of CD8 lymphocytes producing IL-4 (b) and IFN-γ (d) upon polyclonal stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin after a 6-h incubation in controls ( n = 10) and RSV-infected infants ( n = 30). Each symbol characterizes one individual tested. The lines represent medians. The non-parametric Mann–Whitney U -test was used for between-group comparison.

Techniques Used: Incubation, Infection, MANN-WHITNEY

Representative plots of IL-4 and IFN-γ production within the lymphocyte population in an RSV-infected infant (a), control infant (b) and healthy adult (c) upon 6-h whole-blood activation with phorbol 12-myristate 13-acetate (PMA) and ionomycin in the presence of brefeldin A. Numbers indicate the percentage of single or double-stained lymphocytes.
Figure Legend Snippet: Representative plots of IL-4 and IFN-γ production within the lymphocyte population in an RSV-infected infant (a), control infant (b) and healthy adult (c) upon 6-h whole-blood activation with phorbol 12-myristate 13-acetate (PMA) and ionomycin in the presence of brefeldin A. Numbers indicate the percentage of single or double-stained lymphocytes.

Techniques Used: Infection, Activation Assay, Staining

Correlation between IL-4 production within CD4 (a) and CD8 (b) and IFN-γ within CD4 (c) and CD8 (d) among the groups of RSV-infected infants divided according to clinical syndromes: upper respiratory tract infection alone (URTI; n = 7), bronchiolitis (Bronchio; n = 17) and pneumonia (Pneum; n = 6), and controls ( n = 10). Each symbol represents a tested individual. The relationship between variables was assessed by the Spearman rank order coefficient ( r ) and P level value. The lines represent median values.
Figure Legend Snippet: Correlation between IL-4 production within CD4 (a) and CD8 (b) and IFN-γ within CD4 (c) and CD8 (d) among the groups of RSV-infected infants divided according to clinical syndromes: upper respiratory tract infection alone (URTI; n = 7), bronchiolitis (Bronchio; n = 17) and pneumonia (Pneum; n = 6), and controls ( n = 10). Each symbol represents a tested individual. The relationship between variables was assessed by the Spearman rank order coefficient ( r ) and P level value. The lines represent median values.

Techniques Used: Infection

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Enzyme-linked Immunosorbent Assay:

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Activation Assay:

Article Title: Toxoplasma Rhoptry Protein 16 (ROP16) Subverts Host Function by Direct Tyrosine Phosphorylation of STAT6 *
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Recombinant:

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Cell Culture:

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Article Snippet: .. Naive B cells were then cultured in FBS–RPMI and stimulated with mCD154 (1, 2 or 4 U ml−1 ), recombinant human IL-4 (20 ng ml−1 ; R & D Systems) and recombinant human IL-21 (50 ng ml−1 ; R & D Systems) for 24, 48, 72 and 96 h. RNA was extracted from stimulated and unstimulated B cells and used for qRT–PCR analysis. .. qRT–PCR analysis of transcripts Total RNA was extracted from 2–5 × 106 B cells using the RNeasy Mini Kit (Qiagen).

Article Title: Prothymosin ? and a prothymosin ?-derived peptide enhance TH1-type immune responses against defined HER-2/neu epitopes
Article Snippet: .. Monocytes were cultured for 5 days in X-VIVO 15 supplemented with 800 IU/mL recombinant human granulocyte macrophage colony-stimulating factor (GM-CSF) and 500 IU/mL recombinant human IL-4 (both from R & D Systems GmbH, Wiesbaden-Nordenstadt, Germany). .. On day 5, iDCs were treated with LPS (0.5 μg/mL; Sigma-Aldrich), TNF-α (10 ng/mL; R & D Systems), proTα (160 ng/mL) or proαα(100–109) (25 ng/mL) for 1–48 h, concentrations already reported to induce DC maturation [ ].

Quantitative RT-PCR:

Article Title: Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends to modulate antibody class-switch DNA recombination
Article Snippet: .. Naive B cells were then cultured in FBS–RPMI and stimulated with mCD154 (1, 2 or 4 U ml−1 ), recombinant human IL-4 (20 ng ml−1 ; R & D Systems) and recombinant human IL-21 (50 ng ml−1 ; R & D Systems) for 24, 48, 72 and 96 h. RNA was extracted from stimulated and unstimulated B cells and used for qRT–PCR analysis. .. qRT–PCR analysis of transcripts Total RNA was extracted from 2–5 × 106 B cells using the RNeasy Mini Kit (Qiagen).

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    R&D Systems human il 4
    IL-33 promoted anti-RBC antibody production. a PBMCs from AIHA patients (n = 6) were stimulated with anti-IgM, CD40L in the presence of indicated dose of IL-33 for 6d, and then assayed for IgG antibody production. One dot represented the result from one patient; b PBMCs from AIHA patients (n = 5) were stimulated with anti-IgM, CD40L plus IL-33 (20 ng/ml) in the presence of IL-33 neutralizing antibody or control antibody (2 μg/ml) for 6d, and detected for IgG antibody; c – e PBMCs from AIHA patients (n = 4) were stimulated with IL-33 (20 ng/ml) for 72 h, followed by detection for production of <t>IL-4,</t> IL-6 and IL-13 in supernatants
    Human Il 4, supplied by R&D Systems, used in various techniques. Bioz Stars score: 99/100, based on 226 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    IL-33 promoted anti-RBC antibody production. a PBMCs from AIHA patients (n = 6) were stimulated with anti-IgM, CD40L in the presence of indicated dose of IL-33 for 6d, and then assayed for IgG antibody production. One dot represented the result from one patient; b PBMCs from AIHA patients (n = 5) were stimulated with anti-IgM, CD40L plus IL-33 (20 ng/ml) in the presence of IL-33 neutralizing antibody or control antibody (2 μg/ml) for 6d, and detected for IgG antibody; c – e PBMCs from AIHA patients (n = 4) were stimulated with IL-33 (20 ng/ml) for 72 h, followed by detection for production of IL-4, IL-6 and IL-13 in supernatants

    Journal: Journal of Translational Medicine

    Article Title: IL-33 reflects dynamics of disease activity in patients with autoimmune hemolytic anemia by regulating autoantibody production

    doi: 10.1186/s12967-015-0745-0

    Figure Lengend Snippet: IL-33 promoted anti-RBC antibody production. a PBMCs from AIHA patients (n = 6) were stimulated with anti-IgM, CD40L in the presence of indicated dose of IL-33 for 6d, and then assayed for IgG antibody production. One dot represented the result from one patient; b PBMCs from AIHA patients (n = 5) were stimulated with anti-IgM, CD40L plus IL-33 (20 ng/ml) in the presence of IL-33 neutralizing antibody or control antibody (2 μg/ml) for 6d, and detected for IgG antibody; c – e PBMCs from AIHA patients (n = 4) were stimulated with IL-33 (20 ng/ml) for 72 h, followed by detection for production of IL-4, IL-6 and IL-13 in supernatants

    Article Snippet: IL-4, IL-6 and IL-13 levels in culture supernatants were also detected with Human IL-4, IL-6 and IL-13 Quantikine ELISA Kits (R & D Systems) respectively according to the manual’s instructions.

    Techniques:

    STAT6 activation is ROP16-dependent and much faster than IL-4-induced activation. A , parasite infection was synchronized by potassium shift as described under “Experimental Procedures.” Infected HFF cells were fixed 1 min postinvasion with methanol and stained with DAPI and the indicated antibodies. B , HFF cells were stimulated with IL-4 (50 ng/ml), fixed at the indicated time points, and stained with DAPI and the indicated antibodies.

    Journal: The Journal of Biological Chemistry

    Article Title: Toxoplasma Rhoptry Protein 16 (ROP16) Subverts Host Function by Direct Tyrosine Phosphorylation of STAT6 *

    doi: 10.1074/jbc.M110.112359

    Figure Lengend Snippet: STAT6 activation is ROP16-dependent and much faster than IL-4-induced activation. A , parasite infection was synchronized by potassium shift as described under “Experimental Procedures.” Infected HFF cells were fixed 1 min postinvasion with methanol and stained with DAPI and the indicated antibodies. B , HFF cells were stimulated with IL-4 (50 ng/ml), fixed at the indicated time points, and stained with DAPI and the indicated antibodies.

    Article Snippet: To assess the kinetics of IL-4 activation, uninfected cells were treated with serum-free DMEM containing either DMSO or recombinant human IL-4 at 50 ng/ml (R & D Systems, Minneapolis, MN).

    Techniques: Activation Assay, Infection, Staining

    ( a – d ) CD106 (VCAM-1) expression by RA FLSs ( a ), OA FLSs ( b ), dermal fibroblasts ( c ), or IL-4–stimulated dermal fibroblasts ( d ). a – d depict histogram graphs that display the relative red fluorescence intensity of cells stained with anti-CD106 mAb’s (shaded histograms) or a nonspecific isotype control antibody (open histograms). The mean fluorescence intensity ratios of cells stained for CD106 relative to that of control antibody–stained cells are displayed in the upper right-hand corner of each histogram. ( e ) B-cell migration beneath dermal fibroblasts (DFs) is significantly enhanced by IL-4 treatment of dermal fibroblasts. The bars represent the mean (± SD) Nalm-6 or Ramos B-cell migration beneath IL-4–treated dermal fibroblasts relative to the migration beneath untreated dermal fibroblasts, corresponding to 100%. A Significant inhibition of migration with P values

    Journal: Journal of Clinical Investigation

    Article Title: Fibroblast-like synoviocytes support B-cell pseudoemperipolesis via a stromal cell-derived factor-1- and CD106 (VCAM-1)-dependent mechanism

    doi:

    Figure Lengend Snippet: ( a – d ) CD106 (VCAM-1) expression by RA FLSs ( a ), OA FLSs ( b ), dermal fibroblasts ( c ), or IL-4–stimulated dermal fibroblasts ( d ). a – d depict histogram graphs that display the relative red fluorescence intensity of cells stained with anti-CD106 mAb’s (shaded histograms) or a nonspecific isotype control antibody (open histograms). The mean fluorescence intensity ratios of cells stained for CD106 relative to that of control antibody–stained cells are displayed in the upper right-hand corner of each histogram. ( e ) B-cell migration beneath dermal fibroblasts (DFs) is significantly enhanced by IL-4 treatment of dermal fibroblasts. The bars represent the mean (± SD) Nalm-6 or Ramos B-cell migration beneath IL-4–treated dermal fibroblasts relative to the migration beneath untreated dermal fibroblasts, corresponding to 100%. A Significant inhibition of migration with P values

    Article Snippet: Human IL-4 was purchased from R & D Systems Inc. (Minneapolis, Minnesota, USA).

    Techniques: Expressing, Fluorescence, Staining, Migration, Inhibition

    IgE-mediated production and secretion of IL-22 from LAD2 cells. (A) IL-22 was measured by ELISA in the supernatants of LAD2-cell culture stimulated with IgE/SA, IL-4, IL-13, and SEB (n = 3 for each group). (B) IL-22 was measured by ELISA in the supernatants of LAD2-cell culture stimulated in variable concentrations (0.1, 1, or 5 μg/mL) of IgE with SA treatment (n = 3 for each group). (C) Representative double immunostaining (red: tryptase and green: IL-22). Box indicates representative findings (× 200). Scale bar = 50 μm. IL, interleukin; ELISA, enzyme-linked immunosorbent assay; IgE, immunoglobulin E; SEB, Staphylococcal enterotoxin B; SA, streptavidin; Un, unstimulated control.

    Journal: Allergy, Asthma & Immunology Research

    Article Title: Enhanced Type 2 Immune Reactions by Increased IL-22/IL-22Ra1 Signaling in Chronic Rhinosinusitis With Nasal Polyps

    doi: 10.4168/aair.2020.12.6.980

    Figure Lengend Snippet: IgE-mediated production and secretion of IL-22 from LAD2 cells. (A) IL-22 was measured by ELISA in the supernatants of LAD2-cell culture stimulated with IgE/SA, IL-4, IL-13, and SEB (n = 3 for each group). (B) IL-22 was measured by ELISA in the supernatants of LAD2-cell culture stimulated in variable concentrations (0.1, 1, or 5 μg/mL) of IgE with SA treatment (n = 3 for each group). (C) Representative double immunostaining (red: tryptase and green: IL-22). Box indicates representative findings (× 200). Scale bar = 50 μm. IL, interleukin; ELISA, enzyme-linked immunosorbent assay; IgE, immunoglobulin E; SEB, Staphylococcal enterotoxin B; SA, streptavidin; Un, unstimulated control.

    Article Snippet: For TSLP measurement, NHBE cells were stimulated with recombinant human IL-4 (100 ng/mL; R & D Systems), IL-13 (100 ng/mL; R & D Systems), IL-22 (1 or 10 or 100 ng/mL; R & D Systems) for 72 hours.

    Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Double Immunostaining