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: Cell reports

Article Title: TRIM5α Restricts Flavivirus Replication by Targeting the Viral Protease for Proteasomal Degradation

doi: 10.1016/j.celrep.2019.05.040

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Expression plasmids for Langerin (HG13040-UT) and DC-SIGN (HG10200-UT) were purchased from Sino Biological.

Techniques: Transduction, Recombinant, Modification, Protease Inhibitor, Staining, Transfection, Clone Assay, Construct, shRNA, Sequencing, Luciferase, Plasmid Preparation, Expressing, Software, Imaging, Western Blot

Journal: mSphere

Article Title: Term Human Placental Trophoblasts Express SARS-CoV-2 Entry Factors ACE2, TMPRSS2, and Furin

doi: 10.1128/mSphere.00250-21

Figure Lengend Snippet: The ACE2-dependent entry of SARS-CoV-2 pseudovirus into cultured PHT cells. (A) PHT (five donors) and 293T-ACE2 cells inoculated with SARS-CoV-2 protein pseudotyped lentiviruses containing SMNE proteins (spike, membrane, nucleocapsid, and envelope) or MNE proteins, as detailed in Materials and Methods. Cells, harvested on day 2 postinoculation with the virus, were washed three times with PBS and trypsinized to remove adherent virus, and the cell pellet was lysed to release intracellular p24. Shown is p24 content (in picograms per milliliter) in cell lysates. *, P < 0.01 (paired t test). (Inset) 293T cells expressing ACE2, assessed at 2 days or 5 days ( n = 2 for each) and serving as a positive control. (B) PHT were preincubated or not with 20 μg/ml of anti-ACE-2 or anti-DC-SIGN antibody (Ab) and then inoculated with pvSARS-CoV-2 S+. The PHT cells were also inoculated with pseudovirus lacking S protein (pvS-) or with inactivated HIV-1 as negative controls. Cells were harvested on day 2 postinoculation, and p24 content (in picograms per milliliter) was measured in cell lysates, as detailed in Materials and Methods. *, P < 0.05 (ANOVA and Tukey test, n = 5).

Article Snippet: For antibody inhibition experiments, PHT cells were preexposed to 20 μg/ml anti-ACE2 (catalog no. AF933; R&D Systems, Minneapolis, MN ) or anti-DC-SIGN (R&D catalog no. MAB161) for 30 min at 37°C before inoculation with the viruses ( , ).

Techniques: Cell Culture, Expressing, Positive Control