Journal: Cell Communication and Signaling : CCS

Article Title: The transcription factor MITF is a critical regulator of GPNMB expression in dendritic cells

doi: 10.1186/s12964-015-0099-5

Figure Lengend Snippet: PI3K/Akt-inhibition upregulates GPNMB gene expression in human moDC. Immature moDC were generated in vitro with GM-CSF and IL-4 alone (4/GM) or with additional TKI (3 μM imatinib or 3 μM nilotinib) or inhibitors of signal transduction (300 nM Akt inhibitor MK2206 (Akt-inh.), 300 nM Erk inhibitor FR180204 (Erk-inh.), 100 nM PI3K inhibitor LY294002 (PI3K-inh.), 20 nM c-Raf inhibitor 553003 (c-Raf-inh.)) and analyzed for GPNMB expression. Exemplary results from at least three independent experiments using different donors are presented. (A) qRT-PCR analysis: relative level of GPNMB mRNA. The mean (±SD) of duplicate measurements is shown. (B, C) GPNMB protein level of CD209 + moDC (of three different donors) was analyzed by flow cytometry. Where indicated, maturation of moDC was induced by LPS. Data were analyzed using FlowJo software and Difference in Median Fluorescence Intensity (DMFI) of CD209 + cells is shown in the upper right quadrants. (D) Phenotypic changes of immature moDC in the absence (4/GM) or presence of nilotinib or Akt inhibitor were analyzed by flow cytometry. Double stainings were performed with monoclonal antibodies recognizing CD209, CD1a or CD14. DMFI of CD209 + cells is shown in the upper right quadrants.

Article Snippet: After 7 days of culture, if necessary, DC-SIGN + (CD209 + ) moDC were enriched to > 90% purity prior to qRT-PCR and western blot analyses (CD209 MicroBead Kit, Miltenyi).

Techniques: Inhibition, Expressing, Generated, In Vitro, Transduction, Quantitative RT-PCR, Flow Cytometry, Software, Fluorescence

Journal: Cell Communication and Signaling : CCS

Article Title: The transcription factor MITF is a critical regulator of GPNMB expression in dendritic cells

doi: 10.1186/s12964-015-0099-5

Figure Lengend Snippet: The BCR-ABL TKI imatinib and nilotinib or IL-10 inhibit phosphorylation of Akt in human moDC. Western blot analysis of total Akt levels and its phosphorylated form in purified immature CD209 + moDC (of four different donors). moDC were generated in vitro with GM-CSF and IL-4 alone (4/GM) or with additional (A) imatinib (3 μM), (B) nilotinib (3 μM), (C) Akt inhibitor MK2206 (Akt-inh., 300 nM) or (D) IL-10 (10 ng/mL). Indicated time refers to further treatment of cells prior to cell lysis (see ). (E-G) GPNMB protein levels in moDC were analyzed by western blotting. GAPDH served as loading control. Exemplary results from at least three independent experiments using different donors are presented.

Article Snippet: After 7 days of culture, if necessary, DC-SIGN + (CD209 + ) moDC were enriched to > 90% purity prior to qRT-PCR and western blot analyses (CD209 MicroBead Kit, Miltenyi).

Techniques: Western Blot, Purification, Generated, In Vitro, Lysis

Journal: Cell Communication and Signaling : CCS

Article Title: The transcription factor MITF is a critical regulator of GPNMB expression in dendritic cells

doi: 10.1186/s12964-015-0099-5

Figure Lengend Snippet: Imatinib, nilotinib, IL-10 or Akt inhibitor prevent phosphorylation of GSK3ß in human moDC. Western blot analysis of total GSK3ß and GSK3α, as well as their phosphorylated forms in purified immature CD209 + moDC (of four different donors). moDC were generated in vitro with GM-CSF and IL-4 alone (4/GM) or with additional (A) imatinib (3 μM), (B) nilotinib (3 μM), (C) Akt inhibitor MK2206 (Akt-inh., 300 nM) or (D) IL-10 (10 ng/mL). Indicated time refers to further treatment of cells prior to cell lysis (see ). GAPDH served as loading control. Exemplary results from at least three independent experiments using different donors are presented.

Article Snippet: After 7 days of culture, if necessary, DC-SIGN + (CD209 + ) moDC were enriched to > 90% purity prior to qRT-PCR and western blot analyses (CD209 MicroBead Kit, Miltenyi).

Techniques: Western Blot, Purification, Generated, In Vitro, Lysis

Journal: Cell Communication and Signaling : CCS

Article Title: The transcription factor MITF is a critical regulator of GPNMB expression in dendritic cells

doi: 10.1186/s12964-015-0099-5

Figure Lengend Snippet: Upon treatment of moDC with imatinib, nilotinib, IL-10 or MK2206, MITF translocates into the nucleus. Western blot analysis of MITF level and phosphorylation status in the cytoplasmic or nuclear fraction of purified immature CD209 + moDC. moDC were generated in vitro with GM-CSF and IL-4 alone (4/GM) or with (A) imatinib (3 μM), (B) nilotinib (3 μM), (C) Akt inhibitor MK2206 (Akt-inh.; 300 nM) or (D) IL-10 (10 ng/mL). (E) Cells were treated with nilotinib (3 μM). Indicated time refers to further treatment of cells prior to cell lysis (see ). GAPDH served as loading control. Exemplary results from at least three independent experiments using different donors are presented.

Article Snippet: After 7 days of culture, if necessary, DC-SIGN + (CD209 + ) moDC were enriched to > 90% purity prior to qRT-PCR and western blot analyses (CD209 MicroBead Kit, Miltenyi).

Techniques: Western Blot, Purification, Generated, In Vitro, Lysis

Journal: bioRxiv

Article Title: The unique ORF8 protein from SARS-CoV-2 binds to human dendritic cells and induces a hyper-inflammatory cytokine storm

doi: 10.1101/2022.06.06.494969

Figure Lengend Snippet: a) Experimental set up: monocytes were differentiated into immature dendritic cells in presence or absence of ORF8; b) Flow cytometry analysis of the effect of ORF8 on the expression of MHCII, CD80, CD83, CD86, CD40 and CD11c during differentiation of monocytes into immature dendritic cells with IL-4 and GM-CSF compared to only IL-4+GM-CSF differentiated monocytes (blue line); control: monocytes without IL-4+GM-CSF treatment (black line); representative flow cytometry histograms (n=6 donors) c) Flow cytometry analysis of the effect of ORF8 on the expression of DC-SIGN during differentiation of monocytes into immature dendritic cells with IL-4 and GM-CSF (red line) compared to only IL-4+GM-CSF differentiated monocytes (blue line); control: monocytes without IL-4+GM-CSF treatment (black line); representative flow cytometry histograms (n=3 donors) d) Flow cytometry analysis of the effect of the blockade of ORF8 by an inhibitory anti-ORF8 antibody (α-ORF8-iAB) on the expression of DC-SIGN on differentiated dendritic cells compared to only IL-4+GM-CSF differentiated monocytes (blue line) and IL-4+GM-CSF+ORF8 differentiated monocytes (red line); control: monocytes without IL-4+GM-CSF treatment (black line; for isotype control see supplemental ); representative flow cytometry histograms (n=3 donors); e) Immunoprecipitations (IP) of recombinant DC-SIG with 1) no protein control, recombinant ORF8, ORF8 + an inhibitory anti-ORF8 antibody (αORF8), 2) single recombinant proteins (single protein) ORF8 and recombinant human DC-SIGN (positive control) were immunoblotted (IB) with anti-DC-SIGN antibody; * indicates reduced signal intensity compared to ORF8 alone

Article Snippet: For protein-protein interaction, pre-incubated beads were incubated a second time with 2 µg of a recombinant human DC-SIGN protein with a human Fc tag (Sino Biological, Köln, Germany) for 1 h at 4°C with agitation.

Techniques: Flow Cytometry, Expressing, Recombinant, Positive Control

Journal: Cellular and Molecular Life Sciences

Article Title: Glucosylceramide in bunyavirus particles is essential for virus binding to host cells

doi: 10.1007/s00018-023-05103-0

Figure Lengend Snippet: Glucosylceramide (GlcCer) in viral particles promotes Uukuniemi virus (UUKV) binding. A UUKV particles derived from BHK-21 cells in the presence of DL- threo -phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP, 2.5 µM) were bound to freshly seeded naïve BHK-21 cells for 2 h on ice before fixation and western blot analysis with an antibody recognizing the UUKV N protein. B N was semi-quantified from the cells described in A , and the value is presented as a percentage of the N level measured in the sample corresponding to virus binding in the absence of PPMP ( n = 8). C Alternatively, UUKV particles produced in the presence or the absence of PPMP were allowed to bind to BHK-21 cells for 2 h on ice, and binding was assessed by measuring the BHK-21 cell-associated S viral segment by RT-qPCR ( n = 6). D Fluorescently labeled UUKV particles (UUKV-ATTO488) were bound to BHK-21 cells [multiplicity of infection (MOI) ~ 4] on ice for 1 h, and viral binding was evaluated by flow cytometry analysis. E and F BHK-21 cells, A549 human lung epithelial cells, and BHK-21 cells expressing the UUKV receptor DC-SIGN (BHK-21 DC-SIGN +) were preincubated with varying amounts of soluble C6-GlcCer E or C6-Cer F for 2 h and then exposed to UUKV-ATTO488 (MOI ~ 4) on ice for 1 h. Virus binding was measured by flow cytometry, and the data were normalized to those in control samples processed in the absence of soluble C6-GlcCer or C6-Cer. An one-way ANOVA with Dunnett’s multiple comparison test was applied ( n ≥ 3). *, p < 0.05; **, p < 0.01; ns, not significant; RFI, relative fluorescence intensity. G BHK-21 cells were transduced with a retroviral vector system to express DC-SIGN (BHK-21 DC-SIGN +). DC-SIGN expression was measured by flow cytometry analysis using phycoerythrin-conjugated anti-DC-SIGN mAb. H Soluble C6-GlcCer was allowed to bind BHK-21 cells on ice for 2 h before exposure to UUKV for 1 h (MOI ~ 0.5). After virus binding on ice, unbound UUKV particles were washed away, and the cells were incubated at 37 ℃ for 8 h. Infection was quantified by flow cytometry after immunostaining for UUKV N protein. Values are presented as the percentage of the control sample without prebinding of soluble C6-GlcCer ( n = 4)

Article Snippet: The location of DC-SIGN was assessed at the surface of BHK-21 cells (not permeabilized) by flow cytometry using an anti-DC-SIGN phycoerythrin-conjugated antibody (FAB1621P R&D Systems) according to a standard procedure [ ].

Techniques: Virus, Binding Assay, Derivative Assay, Western Blot, Produced, Quantitative RT-PCR, Labeling, Infection, Flow Cytometry, Expressing, Control, Comparison, Fluorescence, Transduction, Retroviral, Plasmid Preparation, Incubation, Immunostaining

Journal: Cells

Article Title: SARS-CoV-2 Spike Protein and Its Receptor Binding Domain Promote a Proinflammatory Activation Profile on Human Dendritic Cells

doi: 10.3390/cells10123279

Figure Lengend Snippet: Dendritic cell activation in response to Spike protein and the RBD is DC-SIGN independent. iDCs were preincubated with an anti-DC-SIGN antibody, stimulated as indicated for 24 h, and then stained and analyzed by flow cytometry. ( A ) CD83 and ( B ) CD86 expression in DCs with the indicated treatment are shown. Graphs show percentage of positivity (left) and GMFI (right) of each marker. Graphs show mean ± SEM; n = 5 different donors.

Article Snippet: Blocking assay was performed incubating iDCs (1 × 10 6 /mL) in complete medium with anti-DC-SIGN antibody (40 μg/mL) (Cat# MAB16211, RD Systems) at 37 °C, for 30 min. After two washes, cells were stimulated as indicated during 24 h and then analyzed.

Techniques: Activation Assay, Staining, Flow Cytometry, Expressing, Marker