Journal: Obesity Facts

Article Title: The Role of MKP-5 in Adipocyte-Macrophage Interactions during Obesity

doi: 10.1159/000505343

Figure Lengend Snippet: MKP-5 overexpression in macrophages is involved in M1-to-M2 macrophage polarization. A 500 μM palmitate (PA) was used to treat Raw264.7 cells, and then IL-6, IL-1β, IL-12, and MKP-5 expression was detected at 1, 3, and 6 h by quantitative real-time (qRT)-PCR. B A stably transfected MKP-5 Raw264.7 cell line was constructed, and MKP-5 overexpression was assessed by qRT-PCR and Western blotting. C Raw-PC and Raw-MKP5 cells were stimulated with the indicated concentration of IFN-γ and LPS for 6 h, and M1 markers were measured by qRT-PCR. D Raw-PC and Raw-MKP5 cells were stimulated using IL-4 for 12 h, and M2 markers were measured by qRT-PCR. E, F With 500 μM PA for 6 h, the expression levels of M1 (E) and M2 markers (F) in Raw-PC and Raw-MKP5 cells were detected by qRT-PCR. Results are means ± SEM of triplicate experiments. EtOH, ethyl alcohol. * p < 0.05.

Article Snippet: Recombinant mouse IL-4 and IFN-γ were obtained from Novus Biologicals (Centennial, CO, USA).

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Stable Transfection, Transfection, Construct, Western Blot, Concentration Assay

Journal: bioRxiv

Article Title: Network analysis reveals a distinct axis of macrophage activation in response to conflicting inflammatory cues

doi: 10.1101/844464

Figure Lengend Snippet: A) Dynamics of predicted gene expression in response to stimulation with LPS+IFNγ or IL4. Stimuli were added at 0 h. B) Kinetics of selected mRNAs in response to stimulation with LPS+IFNγ or IL4. C) mRNA expression profiles predicted by the model, validated against RNA-Seq measurements from peritoneal macrophages treated with LPS+IFNγ or IL4 for 4h. For semi-quantitative comparison between model and experiment, the log2 fold change of each mRNA vs. control was normalized by the root mean square between the M1 and M2 conditions. Classic M1 (orange) and M2 (green) phenotype markers are highlighted.

Article Snippet: Macrophages were assigned to one of three treatment groups: 1) stimulated with 1 μg/mL LPS (Sigma, L2880) and 20 ng/mL IFNγ (R&D, 485-MI) for 4 h; 2) stimulated with 20 ng/mL IL4 (R&D, 404-ML) for 4 h; or 3) untreated for 4 h, serving as the negative control.

Techniques: Gene Expression, Expressing, RNA Sequencing, Comparison, Control

Journal: bioRxiv

Article Title: Network analysis reveals a distinct axis of macrophage activation in response to conflicting inflammatory cues

doi: 10.1101/844464

Figure Lengend Snippet: A) Overall network influence of node knockdowns under stimulation with either LPS+IFNγ (orange) or IL4 (green). Nodes were ranked by the overall influence of their knockdown on all other network nodes, under conditions of LPS+IFNγ stimulation. B) Predicted effect of knockdown of influential nodes on activity of highly sensitive nodes, under conditions of LPS+IFNγ or IL4 treatment.

Article Snippet: Macrophages were assigned to one of three treatment groups: 1) stimulated with 1 μg/mL LPS (Sigma, L2880) and 20 ng/mL IFNγ (R&D, 485-MI) for 4 h; 2) stimulated with 20 ng/mL IL4 (R&D, 404-ML) for 4 h; or 3) untreated for 4 h, serving as the negative control.

Techniques: Knockdown, Activity Assay

Journal: bioRxiv

Article Title: Network analysis reveals a distinct axis of macrophage activation in response to conflicting inflammatory cues

doi: 10.1101/844464

Figure Lengend Snippet: A) Model-predicted signaling module activities in response to IFNγ and IL4 treatments at 4h, column normalized. B) Experimental validation of mRNA expression predicted in response to IFNγ, IL4, or IFNγ+IL4. For both experimental data and model predictions, mRNA were independently normalized by RMS-normalized log2 fold change at 4 h. C) Predicted expression dynamics of selected mRNAs in response to IFNγ, IL4, or IFNγ+IL4. D) Context-dependent network response to node knockdowns under treatments of IFNγ, IL4, or IFNγ+IL4.

Article Snippet: Macrophages were assigned to one of three treatment groups: 1) stimulated with 1 μg/mL LPS (Sigma, L2880) and 20 ng/mL IFNγ (R&D, 485-MI) for 4 h; 2) stimulated with 20 ng/mL IL4 (R&D, 404-ML) for 4 h; or 3) untreated for 4 h, serving as the negative control.

Techniques: Biomarker Discovery, Expressing

Journal: Scientific Reports

Article Title: Parkinsonian neurotoxicants impair the anti-inflammatory response induced by IL4 in glial cells: involvement of the CD200-CD200R1 ligand-receptor pair

doi: 10.1038/s41598-020-67649-4

Figure Lengend Snippet: Effect of MPP+ treatment on the mRNA expression of anti-inflammatory markers. IL10, TGFβ, IL1ra, Arg1, MR, Fizz1 and Ym1 mRNA expression in primary ( a ) mixed glial cultures and ( b ) microglial cultures treated for 24 h with 10 or 25 μM MPP+, in the absence and presence of IL4 (50 ng/mL). Genes encoding Rn18s and β-actin were used as the reference genes. Bars correspond to the means + SEM of four independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 vs control (C); # p < 0.05, ## p < 0.01 and ### p < 0.001 vs IL4; one-way ANOVA and Newman-Keuls post-hoc test. & p < 0.05, && p < 0.01 and &&& p < 0.001 vs C, one-way ANOVA and Newman-Keuls post-hoc test considering only the IL4-free groups, which was performed to detect whether the high values observed in the IL4 group hindered the detection of a statistical significance for the effects of MPP+ alone.

Article Snippet: Recombinant mouse IL4 expressed in CHO cells was used (Creative BioMart, Shirley, NY, USA).

Techniques: Expressing, Control

Journal: Scientific Reports

Article Title: Parkinsonian neurotoxicants impair the anti-inflammatory response induced by IL4 in glial cells: involvement of the CD200-CD200R1 ligand-receptor pair

doi: 10.1038/s41598-020-67649-4

Figure Lengend Snippet: Effect of rotenone treatment on the mRNA expression of anti-inflammatory markers. IL10, TGFβ, IL1ra, Arg1, MR, Fizz1 and Ym1 mRNA expression in primary ( a ) mixed glial cultures and ( b ) microglial cultures treated for 24 h with 40 or 100 nM rotenone (Rot), in the absence and presence of IL4 (50 ng/mL). Genes encoding Rn18s and β-actin were used as the reference genes. Bars correspond to the means+ SEM of four independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 vs control (C); # p < 0.05, ## p < 0.01 and ### p < 0.001 vs IL4; one-way ANOVA and Newman-Keuls post-hoc test. &&& p < 0.001 vs C, one-way ANOVA and Newman-Keuls post-hoc test considering only the IL4-free groups, which was performed to detect whether the high values observed in the IL4 group hindered the detection of a statistical significance of the effects of Rot alone.

Article Snippet: Recombinant mouse IL4 expressed in CHO cells was used (Creative BioMart, Shirley, NY, USA).

Techniques: Expressing, Control

Journal: Scientific Reports

Article Title: Parkinsonian neurotoxicants impair the anti-inflammatory response induced by IL4 in glial cells: involvement of the CD200-CD200R1 ligand-receptor pair

doi: 10.1038/s41598-020-67649-4

Figure Lengend Snippet: ARG1 and MR protein levels in primary glial cell cultures treated with MPP+ or rotenone. ARG1 and MR levels were determined in the total protein extracts from ( a , c ) mixed glial cultures and ( b , d ) microglial cultures treated with 10 or 25 μM MPP+ or 40 or 100 nM rotenone (Rot) for 24 h, in the absence or presence of IL4 (50 ng/mL). Images of representative western blots are presented. Bars correspond to the means+ SEM of four independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 vs control (C); #p < 0.05, ## p < 0.01 and ### p < 0.001 vs IL4; one-way ANOVA and Newman-Keuls post-hoc test.

Article Snippet: Recombinant mouse IL4 expressed in CHO cells was used (Creative BioMart, Shirley, NY, USA).

Techniques: Western Blot, Control

Journal: Scientific Reports

Article Title: Parkinsonian neurotoxicants impair the anti-inflammatory response induced by IL4 in glial cells: involvement of the CD200-CD200R1 ligand-receptor pair

doi: 10.1038/s41598-020-67649-4

Figure Lengend Snippet: Effect of MPP+ or rotenone treatment on CD200 and CD200R1 mRNA expression. CD200full, CD200tr and CD200R1 mRNA expression in primary mixed glial cultures ( a , c ) and microglial cultures ( b , d ) treated for 24 h with 10 or 25 μM MPP+ ( a , b ) or 40 or 100 nM rotenone (Rot) ( c , d ), both in the absence and presence of IL4 (50 ng/mL). Genes encoding Rn18s and β-actin were used as the reference genes. Bars correspond to the means + SEM of four independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 vs control (C); ###p < 0.001 vs IL4; one-way ANOVA and Newman-Keuls post-hoc test.

Article Snippet: Recombinant mouse IL4 expressed in CHO cells was used (Creative BioMart, Shirley, NY, USA).

Techniques: Expressing, Control

Journal: Scientific Reports

Article Title: Parkinsonian neurotoxicants impair the anti-inflammatory response induced by IL4 in glial cells: involvement of the CD200-CD200R1 ligand-receptor pair

doi: 10.1038/s41598-020-67649-4

Figure Lengend Snippet: Phagocytosis in microglial cells exposed to MPP+ or rotenone. The phagocytosis of fluorescent microspheres was tested in primary microglial cultures treated with 10 or 25 μM MPP+ or 40 or 100 nM rotenone (Rot) in the absence and presence of IL4 (50 ng/mL). Intracellular microspheres were quantified after the immunofluorescence labeling of microglial cells using an anti-Iba1 antibody. ( a ) Classification of microglial cells into phagocytic (cells containing microspheres) or non-phagocytic (cells without microspheres) in the different experimental conditions. Bars correspond to percentages of the total amount of cells of 5 independent cultures for each experimental condition. p < 0.001 in the MPP+ and the rotenone experiments, Chi-square test; ***p < 0.001 vs C, ###p < 0.001 vs IL4, and &&& p < 0.001 vs MPP+ or Rot alone; Chi-square tests. ( b ) Means of fluorescent microspheres per cell in phagocytic cells. Bars correspond to the means + SEM of 5 independent experiments. *p < 0.05 and **p < 0.01 vs. control (C); one-way ANOVA and Newman-Keuls post-hoc test. ( c ) Classification of phagocytic microglial cells into three groups by their degree of phagocytic activity: cells with fewer microspheres/cell than the mean value minus the variance in control cells, cells with microspheres/cell in the range of the mean value ± variance in control cells and cells with microspheres/cell higher than the mean value plus the variance in control cells. Bars correspond to percentages of the total amount of cells of 5 independent cultures for each experimental condition. p < 0.001 in the MPP+ and the rotenone experiments, Chi-square test; ***p < 0.001 vs C, # p < 0.05 and ### p < 0.001 vs IL4, and & p < 0.05 vs MPP+ or Rot alone; Chi-square tests.

Article Snippet: Recombinant mouse IL4 expressed in CHO cells was used (Creative BioMart, Shirley, NY, USA).

Techniques: Immunofluorescence, Labeling, Control, Activity Assay

Journal: Scientific Reports

Article Title: Parkinsonian neurotoxicants impair the anti-inflammatory response induced by IL4 in glial cells: involvement of the CD200-CD200R1 ligand-receptor pair

doi: 10.1038/s41598-020-67649-4

Figure Lengend Snippet: Metabolic activity and intracellular ATP levels in primary glial cell cultures treated with MPP+ or rotenone. ( a – d ) MTT reduction in mixed glial cultures (left column) and microglial cultures (right column) treated for 24 h with 10 or 25 μM MPP+ ( a , b ) or 40 or 100 nM Rot ( c , d ) in the absence and presence of IL4 (50 ng/mL). ( e – h ) Intracellular ATP levels in mixed glial cultures (left column) and microglial cultures (right column) treated with 10 or 25 μM MPP+ ( e , f ) or 40 or 100 nM rotenone (Rot) ( g , h ) for 24 h, in the absence and presence of IL4 (50 ng/mL). Bars correspond to the means + SEM of five independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 vs control (C); ## p < 0.01 and ### p < 0.001 vs IL4; one-way ANOVA and Newman-Keuls post-hoc test.

Article Snippet: Recombinant mouse IL4 expressed in CHO cells was used (Creative BioMart, Shirley, NY, USA).

Techniques: Activity Assay, Control

Journal: Scientific Reports

Article Title: Parkinsonian neurotoxicants impair the anti-inflammatory response induced by IL4 in glial cells: involvement of the CD200-CD200R1 ligand-receptor pair

doi: 10.1038/s41598-020-67649-4

Figure Lengend Snippet: Effect of MPP+ or rotenone treatment on the expression of genes involved in regulating energy metabolism in microglial cultures. ( a, b ) Pgc1β, ( c , d ) Carkl, ( e , f ) glucose transporter (Glut)1, ( g , h ) Pfkp and ( i , j) Hif1α mRNA expression in microglial cultures treated for 24 h with 10 or 25 μM MPP+ (left column) or 40 or 100 nM rotenone (Rot, right column), in the absence and presence of IL4 (50 ng/mL). Genes encoding Rn18s and β-actin were used as the reference genes. Bars correspond to the means + SEM of four independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 vs control (C); ##p < 0.01 and ###p < 0.001 vs IL4; one-way ANOVA and Newman-Keuls post-hoc test.

Article Snippet: Recombinant mouse IL4 expressed in CHO cells was used (Creative BioMart, Shirley, NY, USA).

Techniques: Expressing, Control

Journal: Scientific Reports

Article Title: Parkinsonian neurotoxicants impair the anti-inflammatory response induced by IL4 in glial cells: involvement of the CD200-CD200R1 ligand-receptor pair

doi: 10.1038/s41598-020-67649-4

Figure Lengend Snippet: Effect of MPP+ or rotenone treatment on the expression of genes involved in regulating energy metabolism in mixed glial cultures. ( a , b ) Pgc1β, ( c , d ) Carkl, ( e , f ) the glucose transporter (Glut)1, ( g , h ) Pfkp and ( i , j ) Hif1α mRNA expression in mixed glial cultures treated for 24 h with 10 or 25 μM MPP+ (left column) or 40 or 100 nM rotenone (Rot, right column), in the absence and presence of IL4 (50 ng/mL). Genes encoding Rn18s and β-actin were used as the reference genes. Bars correspond to the means + SEM of four independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 vs control (C); ## p < 0.01 and ### p < 0.001 vs IL4; one-way ANOVA and Newman-Keuls post-hoc test.

Article Snippet: Recombinant mouse IL4 expressed in CHO cells was used (Creative BioMart, Shirley, NY, USA).

Techniques: Expressing, Control

Journal: Scientific Reports

Article Title: Parkinsonian neurotoxicants impair the anti-inflammatory response induced by IL4 in glial cells: involvement of the CD200-CD200R1 ligand-receptor pair

doi: 10.1038/s41598-020-67649-4

Figure Lengend Snippet: Lactate release in primary glial cell cultures treated with MPP+ or rotenone. Extracellular lactate levels in mixed glial cultures (left column) and microglial cultures (right column) treated with 10 μM MPP+ ( a , b ) or 40 nM rotenone (Rot) ( c , d ) for 24 h, in the absence and presence of IL4 (50 ng/mL). Bars correspond to the means + SEM of three independent experiments. *p < 0.05 vs control (C); one-way ANOVA and Newman-Keuls post-hoc test.

Article Snippet: Recombinant mouse IL4 expressed in CHO cells was used (Creative BioMart, Shirley, NY, USA).

Techniques: Control

Journal: Scientific Reports

Article Title: Parkinsonian neurotoxicants impair the anti-inflammatory response induced by IL4 in glial cells: involvement of the CD200-CD200R1 ligand-receptor pair

doi: 10.1038/s41598-020-67649-4

Figure Lengend Snippet: Summary of the effects of MPP+ and rotenone exposure on IL4-induced immune response on primary glial cell cultures. Immunometabolism refers to the close relationship existing between immune response and cell metabolism and the hypothesis that metabolic reprogramming is behind the development of a proper immune response by glial cells. IL4 treatment induces the expression of anti-inflammatory markers and CD200R1, and the stimulation of the CD200R1 immune receptor in microglial cells may play a role in the former by a still underexplored mechanism (right side of the figure, green and orange arrows). MPP+ and rotenone are inhibitors of the mitochondrial respiratory chain and consequently impair oxidative phosphorylation, which is associated to changes in the expression of genes encoding proteins involved in the control of glial cell metabolism in both microglial (µG) and mixed glial (MG) cultures (left side of the figure, red arrows). The inhibition of oxidative phosphorylation by the neurotoxicants stimulates aerobic glycolysis (associated to lactate production) and the pentose phosphate pathway in mixed glial cultures, while fatty acid oxidation and not the pentose phosphate pathway is stimulated in microglial cell cultures. This impairment may be responsible for the inhibition of the expression of anti-inflammatory markers observed in mixed glial cultures and, to a lesser extent, in microglial cultures after MPP+ and rotenone treatment. CARKL sedoheptulokinase; GLUT1 glucose transporter 1; HIF1α hypoxia-inducible factor 1, alpha subunit; PFKP phosphofructokinase, platelet; PGC1β peroxisome proliferator-activated receptor gamma coactivator 1, beta.

Article Snippet: Recombinant mouse IL4 expressed in CHO cells was used (Creative BioMart, Shirley, NY, USA).

Techniques: Expressing, Phospho-proteomics, Control, Inhibition

Journal: PLoS ONE

Article Title: IL-4 Suppresses the Responses to TLR7 and TLR9 Stimulation and Increases the Permissiveness to Retroviral Infection of Murine Conventional Dendritic Cells

doi: 10.1371/journal.pone.0087668

Figure Lengend Snippet: We treated cDCs at day 6-7 of culture with IL-4 or left them untreated for 24 h. We then stimulated the cDCs with CpG 1826 (10 ug/ml) or R848 (1 ug/ml) for 6 h and then analyzed by qPCR the expression of the IFN-responsive genes ISG15 and Mx-1. All of the conditions were normalized against the control (untreated DCs in medium only) in each experiment. Results are average of three independent experiments, performed with three independent bone marrow-derived cultures from 3 different mice; * p <0.05; ** p <0.01.

Article Snippet: Resting DC cultures at day 6 or 7 were treated for 24 h with recombinant mouse IL-4 at 2.5 ng/ml (BD Biosciences), a dose we have previously found efficient to affect cDCs .

Techniques: Expressing, Derivative Assay

Journal: PLoS ONE

Article Title: IL-4 Suppresses the Responses to TLR7 and TLR9 Stimulation and Increases the Permissiveness to Retroviral Infection of Murine Conventional Dendritic Cells

doi: 10.1371/journal.pone.0087668

Figure Lengend Snippet: A. We analyzed the gene expression of IRF7 by qPCR after stimulation with CpG 1826 (10 ug/ml) or R848 (1 ug/ml) for 6 h in cDCs treated with IL-4 or left untreated for 24 h. All of the conditions were normalized against the control (untreated DCs in medium only) in each experiment. Results are the average of three independent experiments; * p <0.05; ** p <0.01. B. We analyzed the IRF7 protein expression by Western blotting in cDCs 8 h after stimulation with CpG or R848. Bar graph represents the ratio of integrated density values of IRF7 vs GAPDH (loading control). One representative blot is shown from testing five independent cultures.

Article Snippet: Resting DC cultures at day 6 or 7 were treated for 24 h with recombinant mouse IL-4 at 2.5 ng/ml (BD Biosciences), a dose we have previously found efficient to affect cDCs .

Techniques: Expressing, Western Blot

Journal: PLoS ONE

Article Title: IL-4 Suppresses the Responses to TLR7 and TLR9 Stimulation and Increases the Permissiveness to Retroviral Infection of Murine Conventional Dendritic Cells

doi: 10.1371/journal.pone.0087668

Figure Lengend Snippet: A . We treated cDCs with IL-4 or left them untreated for 24 h. We then stimulated the cDCs with CpG or R848 for 24 h and then analyzed MHC class I expression. Histogram bars represent averages and standard errors (SE) of the median fluorescence intensity (MdFI) of seven experiments conducted with seven independent cDC cultures; *p<0.05,**p<0.01. B . Western Blot analyses of STAT2 and STAT1 expression in cDCs treated or not IL-4 for 24 h and then stimulated for 8 h with 10 ug/ml of CpG 1826 or 1 ug/ml of R848. Actin was used as loading control. We show a representative blot of three independent experiments. Bar graph represents the ratio of integrated density values of STAT1 or STAT2 vs Actin (loading control).

Article Snippet: Resting DC cultures at day 6 or 7 were treated for 24 h with recombinant mouse IL-4 at 2.5 ng/ml (BD Biosciences), a dose we have previously found efficient to affect cDCs .

Techniques: Expressing, Fluorescence, Western Blot

Journal: PLoS ONE

Article Title: IL-4 Suppresses the Responses to TLR7 and TLR9 Stimulation and Increases the Permissiveness to Retroviral Infection of Murine Conventional Dendritic Cells

doi: 10.1371/journal.pone.0087668

Figure Lengend Snippet: A. We analyzed the gene expression of IL-6, TNFα, IL-12p35 and IL-12p40 by qPCR after stimulation with CpG or R848 for 6 h in cDCs treated or not with IL-4 for 24 h. Averages and SE of four independent cDC cultures are shown (* p <0.05; ** p <0.01, *** p <0.001). B. We measured by ELISA the levels of IL-6, TNFα and IL-12p70 in the supernatants of DC cultures treated or not with IL-4 for 24 h and then stimulated with R848 or CpG 1826 for 24 h. Averages and SE of four independent cultures are shown.

Article Snippet: Resting DC cultures at day 6 or 7 were treated for 24 h with recombinant mouse IL-4 at 2.5 ng/ml (BD Biosciences), a dose we have previously found efficient to affect cDCs .

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: IL-4 Suppresses the Responses to TLR7 and TLR9 Stimulation and Increases the Permissiveness to Retroviral Infection of Murine Conventional Dendritic Cells

doi: 10.1371/journal.pone.0087668

Figure Lengend Snippet: A. We analyzed by qPCR the gene expression of the IFN-responsive genes IRF7, ISG15 and Mx-1 induced by 6 h stimulation with CpG or R848 in wild type (WT) (C57BL/6) and STAT6-KO cDCs treated or not for 24 h with IL-4. Results were normalized to WT control cells not treated with IL-4. B. We analyzed the MHC Class I expression on cDCs by flow cytometry. Results are shown as median fluorescence intensity (MdFI). Averages and SE of three independent BMDC cultures are shown. (* p<0.05,**p<0.01, ***p<0.0001). C. Western Blot analyses of STAT2 expression in cDCs treated or not for 24 h with IL-4 and harvested 8 h after stimulation with CpG or R848 in WT and STAT6-KO BMDCs. GAPDH was used as loading control. One blot representative of three independent experiments is shown. Numbers below the blot represent the percentage of the normalized integrated density values against GAPDH (loading control).

Article Snippet: Resting DC cultures at day 6 or 7 were treated for 24 h with recombinant mouse IL-4 at 2.5 ng/ml (BD Biosciences), a dose we have previously found efficient to affect cDCs .

Techniques: Expressing, Flow Cytometry, Fluorescence, Western Blot

Journal: PLoS ONE

Article Title: IL-4 Suppresses the Responses to TLR7 and TLR9 Stimulation and Increases the Permissiveness to Retroviral Infection of Murine Conventional Dendritic Cells

doi: 10.1371/journal.pone.0087668

Figure Lengend Snippet: We measured by ELISA the levels of IL-6 and IL-12p70 in the supernatants of cDC cultures from wild type and STAT6-KO mice treated or not for 24 h with IL-4 and then stimulated with CpG or R848 for 6 h (IL-6) or 48 h (IL-12p70). Averages and SE of three independent cultures are shown (* p<0.05).

Article Snippet: Resting DC cultures at day 6 or 7 were treated for 24 h with recombinant mouse IL-4 at 2.5 ng/ml (BD Biosciences), a dose we have previously found efficient to affect cDCs .

Techniques: Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: IL-4 Suppresses the Responses to TLR7 and TLR9 Stimulation and Increases the Permissiveness to Retroviral Infection of Murine Conventional Dendritic Cells

doi: 10.1371/journal.pone.0087668

Figure Lengend Snippet: A. We analyzed by qPCR the gene expression of TLR7 and TLR9 in cDCs treated or not with IL-4 for 24 h. Graphs show averages and SE of six independent cultures. B. We analyzed the protein expression of TLR7 in the total lysates of cDC treated or not for 24 h with IL-4. C. We analyzed by immunofluorescence staining TLR7 expression in cDCs after 24 h treatment with IL-4. The nucleus was stained with DAPI and TLR7 was stained using a rabbit polyclonal anti-body to TLR7 and a secondary anti-rabbit IgG conjugated to Cy3. A minimum of five fields in each slide was read for each condition at a magnification of 40×. Picture representative of five independent cultures.

Article Snippet: Resting DC cultures at day 6 or 7 were treated for 24 h with recombinant mouse IL-4 at 2.5 ng/ml (BD Biosciences), a dose we have previously found efficient to affect cDCs .

Techniques: Expressing, Immunofluorescence, Staining

Journal: PLoS ONE

Article Title: IL-4 Suppresses the Responses to TLR7 and TLR9 Stimulation and Increases the Permissiveness to Retroviral Infection of Murine Conventional Dendritic Cells

doi: 10.1371/journal.pone.0087668

Figure Lengend Snippet: A. We analyzed the gene expression of SOCS1, -2 and -3 in cDCs by qPCR after 24 h treatment with IL-4. Results were normalized against untreated cells. Graphs show averages and SE of 6 independent cultures. B. We analyzed the protein expression of SOCS2 in the total cDC lysates after 24 h treatment with IL-4. Results were normalized against GAPDH. C . We analyzed the protein expression of SOCS2 in wild-type (WT) and STAT6-KO cDCs that had been treated or not with IL-4 for 24 h and then stimulated with CpG or R848 for 8 h. Results were normalized against GAPDH. Blot representative of three sets of experiments.

Article Snippet: Resting DC cultures at day 6 or 7 were treated for 24 h with recombinant mouse IL-4 at 2.5 ng/ml (BD Biosciences), a dose we have previously found efficient to affect cDCs .

Techniques: Expressing

Journal: PLoS ONE

Article Title: IL-4 Suppresses the Responses to TLR7 and TLR9 Stimulation and Increases the Permissiveness to Retroviral Infection of Murine Conventional Dendritic Cells

doi: 10.1371/journal.pone.0087668

Figure Lengend Snippet: We analyzed the expression of IFN-responsive genes induced by CpG or R848 stimulation in splenic DCs. We cultured the total splenocytes from C57BL/6 mice as 30–50 million cells in 3 ml each well in GM-CSF-complete medium and 2.5 ng/ml of IL-4 overnight and then stimulated with 50 ug CpG or 5 ug R848 for 4 h. Subsequently we sorted the CD11c+ DCs using Miltenyi magnetic bead separation. All the conditions were normalized against the control (GM-CSF medium only) in each experiment. Line graphs show the response calculated as fold change from no IL-4 control of three independent experiments (* p <0.05).

Article Snippet: Resting DC cultures at day 6 or 7 were treated for 24 h with recombinant mouse IL-4 at 2.5 ng/ml (BD Biosciences), a dose we have previously found efficient to affect cDCs .

Techniques: Expressing, Cell Culture