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