Journal: The Journal of Biological Chemistry

Article Title: Interferon-? and Granulocyte/Monocyte Colony-stimulating Factor Production by Natural Killer Cells Involves Different Signaling Pathways and the Adaptor Stimulator of Interferon Genes (STING)

doi: 10.1074/jbc.M112.435602

Figure Lengend Snippet: A, both accessory cytokines IL-15/IL-18 and CpG-ODN are necessary for NF-κB p65 nuclear translocation. Purified NK cells were primed overnight with IL-15 and IL-18 (10 ng/ml each) and then challenged with CpG-ODN (1 μm). Nuclear translocation of p65 was measured by immunofluorescence after 60-min stimulation. Hoeschst was used to stain the nucleus. B, to quantify the results shown in A, the percentage of nuclear translocation was determined by counting 100 cells. The figure is representative of three independent experiments. ***, p < 0.001 versus unstimulated cells (control) using one-way ANOVA and Fisher least significance difference test.

Article Snippet: The murine recombinant cytokines (IL-2, IL-15) were from Peprotech, and IL-18 was from Medical and Biological Laboratories.

Techniques: Translocation Assay, Purification, Immunofluorescence, Staining

Journal: The Journal of Biological Chemistry

Article Title: Interferon-? and Granulocyte/Monocyte Colony-stimulating Factor Production by Natural Killer Cells Involves Different Signaling Pathways and the Adaptor Stimulator of Interferon Genes (STING)

doi: 10.1074/jbc.M112.435602

Figure Lengend Snippet: Purified spleen NK cells from WT and mice deficient for MyD88, TLR9, STING, TRIF, UNC93b1, IFN-α/βR, and IL-12RB2 were stimulated with accessory cytokines (IL-15 and IL-18 at 10 ng/ml each) and CpG-ODN (1 μm) for 48 h, and IFN-γ (A) and GM-CSF (B) were measured in the supernatants by ELISA. The MyD88 inhibitory peptide (MyD-Pep) was also applied on the sting−/− cells at 25 μm. Data are mean ± S.E. of five experiments. *, p < 0.05; *, p < 0.01; ***, p < 0.001 versus WT using one-way ANOVA and Fisher least significance difference test. C, purified NK cells were primed overnight with IL-15 and IL-18 (10 ng/ml each) and challenged with CpG-ODN (1 μm) in presence of GolgiStop. IL-12p40/70 was then stained intracellularly using specific antibodies and detected on the gated NKp46+ cells. The left histogram shows the IL-12p40/70 stain as percentage of positive cells, the middle graph shows the mean fluorescence intensity (MFI), and the right histograms show the expression of IL-12Rβ2 on the NK cells cultured in the same conditions. Data are the mean ± S.E. of five experiments. ***, p < 0.001 versus cells without stimulation (control) using one-way ANOVA and Fisher least significance difference test. D, purified NK cells were stimulated with IL-15/IL-18 and CpG-ODN labeled with A488. After 60 min, the cells were fixed, stained with an anti-STING antibody, and processed for confocal microscopy. The nucleus is visualized with Hoechst. Arrows indicate spots where an proximally overlapping was observed for cytosolic STING and speckled CpG-ODN stain.

Article Snippet: The murine recombinant cytokines (IL-2, IL-15) were from Peprotech, and IL-18 was from Medical and Biological Laboratories.

Techniques: Purification, Enzyme-linked Immunosorbent Assay, Staining, Fluorescence, Expressing, Cell Culture, Labeling, Confocal Microscopy

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Syk is indispensable for CpG-induced activation and differentiation of human B cells

doi: 10.1007/s00018-014-1806-x

Figure Lengend Snippet: Syk regulates CpG-driven B cell activation, proliferation and cytokine production (a) 2 × 105 resting tonsillar B cells/well were activated with a suboptimal concentration of CpG (0.5 µg/ml) in the presence or absence of Syk or Src inhibitors (iSykIV, iSykII or src inhibitor). As control, cells were cultured in medium or with non-activating GpC ODN. Cells were harvested after pulsing with 1 μCi/well H3-thymidine for the last 18 h of culture. Results are expressed as normalized mean cpm ± SD of triplicate samples of one representative experiment of 5. b, c 2 × 105 resting tonsillar B cells/well were activated with 0.5 µg/ml CpG in the presence or absence of different concentrations of Syk inhibitor IV (iSyk IV), as indicated. b After 48 h, changes in the expression of CD80, CD86 and CD40 were investigated by flow cytometry. Expression of CD80, CD86 or CD40 is illustrated as flow cytometric histograms as well as by diagrams showing normalized mean relative MFI ± SD of duplicate samples. One representative experiment of 3 is shown. c Secretion of IL-10, IL-6 and TNF-α was assessed after the stimulation of resting tonsillar B cells with CpG for 48 h. Results of the Flow Cytomix assay show the amount of secreted cytokines of pooled duplicate samples. One representative experiment of 3 is shown. d Expression of Syk in B cells after transfection with 1 µM (red solid line) or 2 µM (red dotted line) Syk-targeting siRNA. As control, non-transfected (black solid line) or 2 µM control siRNA treated (dotted black line) B cells were used. Histograms are representative of 3 independent experiments. e 1 × 106 B cells/sample transfected with Syk siRNA or control siRNA were left untreated or activated with 0.5 µg/ml CpG. Proliferation of B cells was measured by CFSE and results are illustrated as mean % of proliferated cells ± SD of duplicate samples. IL-6 and IL-10 production were assessed by FlowCytomix assay and data show the amount of secreted cytokines of pooled duplicate samples. Results are representative of 3 independent experiments

Article Snippet: Recombinant cytokines (IL-2 and IL-10) were purchased from Immunotools.

Techniques: Activation Assay, Concentration Assay, Cell Culture, Expressing, Flow Cytometry, Transfection