Journal: Cell stem cell

Article Title: The Mitochondrial Transacylase, Tafazzin, Regulates AML Stemness by Modulating Intracellular Levels of Phospholipids

doi: 10.1016/j.stem.2019.02.020

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: CL075 , TOCRIS , Cat# 6142.

Techniques: Recombinant, Modification, Western Blot, Binding Assay, XF Assay, Electron Microscopy, Staining, High Performance Thin Layer Chromatography, Reverse Transcription, Plasmid Preparation, Apoptosis Assay, DC Protein Assay, SYBR Green Assay, Gene Expression, shRNA, Control, CRISPR, Software

Journal: Journal of leukocyte biology

Article Title: TLR8 Ligation Induces Apoptosis of Monocytic Myeloid-Derived Suppressor Cells

doi: 10.1002/JLB.5AB0217-070R

Figure Lengend Snippet: mRNA expression of TLR8 (A), TLR7 (B) and TLR9 (C) on mDC, pDC, mMDSC and gMDSC in three donors. Columns and bars represents mean (± SE) of duplicate measurements. mMDSC, white column; other cell types, black columns.

Article Snippet: In some assays, the cells were cultured with 0.5uM and 2uM of TLR7/8 agonist CL-075, 10uM and 50uM of TLR7 agonist Imiquimod, and 50nM and 100nM of TLR 9 agonist CpG ODN2006 with PBS control (all InvivoGen).

Techniques: Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Endosomal localization of TLR8 confers distinctive proteolytic processing on human myeloid cells.

doi: 10.4049/jimmunol.1401375

Figure Lengend Snippet: FIGURE 1. The flexible loop between LRR14 and LRR15 was required for CL075-induced TLR8-mediated NF-kB activation in HEK293 cells. (A) HEK293 cells were transiently transfected with vector alone or maximum amounts of C-terminal FLAG-tagged wild-type or mutant TLR8 plasmids (TLR8Dloop and TLR8-C), together with NF-kB–luciferase reporter plasmid and phRL-TK. Twenty-four hours after transfection, cells were stimulated with CL075 (2.5 mg/ml), DOTAP alone, or ssRNA40 complexed with DOTAP (2.5, 5, or 10 mg/ml) or were left untreated. Luciferase activity was measured 12 h after stimulation and expressed as fold induction relative to the activity of unstimulated cells. Representative data from three independent experiments, each performed in triplicate, are shown (mean 6 SD). (B) Protein expression of wild-type and mutant TLR8 in HEK293 cells. Cell lysates prepared in (A) were subjected to SDS-PAGE (7.5%), followed by Western blotting with anti-FLAG mAb and anti–tubulin-a mAb. (C) Coexpression of UNC93B1 promoted CL075-induced TLR8-mediated NF-kB activation in HEK293 cells. HEK293 cells were transfected with the indicated plasmids together with NF-kB–luciferase reporter plasmid and phRL-TK. Cells were stimulated with 2.5 mg/ml CL075, DOTAP alone, or ssRNA40 complexed to DOTAP or were left untreated. Luciferase activity was measured 12 h after stimulation and expressed as fold induction relative to the activity of unstimulated cells. (D) Cell lysates prepared in (C) were subjected to SDS-PAGE (7.5%), followed by Western blotting with anti-FLAG mAb, anti–TLR8-N mAb, and anti–tubulin-a mAb. Filled arrowheads indicate full-length TLR8. Open arrowheads indicate N-terminal half of TLR8. Arrow indicates C-terminal half of TLR8.

Article Snippet: Monocyte-derived macrophages (5 3 105 /ml) were pretreated with DC1 (20 mM) or DMSO for 4 h and then were stimulated with CL075 (2.5 mg/ml), ssRNA40 complexed to DOTAP (2.5 mg/ml), or LPS (1 mg/ml) or left untreated in the presence of inhibitors for another 24 h. To examine stepwise processing of TLR8-N, monocytes were treated with 10 mM z-FA-FMK in the presence of 20 ng/ml recombinant hGM-CSF for 24 or 48 h and then stimulated with indicated ligands for 24 h. IL-12p40 in culture supernatants was measured by ELISA (R&D Systems).

Techniques: Activation Assay, Transfection, Plasmid Preparation, Mutagenesis, Luciferase, Activity Assay, Expressing, SDS Page, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Endosomal localization of TLR8 confers distinctive proteolytic processing on human myeloid cells.

doi: 10.4049/jimmunol.1401375

Figure Lengend Snippet: FIGURE 2. Human TLR8 underwent proteolytic processing in IFN-g–treated THP-1 cells. (A) THP-1 cells (5 3 105/ml) were stimulated with 20 ng/ml IFN-g or were left untreated for 15 h. Expression of TLR7, TLR8, and TLR9 mRNAs was analyzed by RT-PCR using specific primers (Supplemental Table I) (56). (B) Lysates of IFN-g2treated or untreated THP-1 cells were subjected to SDS-PAGE, followed by Western blotting with anti–hTLR8-C pAb and anti– tubulin-a mAb. Arrowhead indicates full-length TLR8. Arrow indicates C-terminal half of TLR8. (C) Control or TLR8–knocked down IFN-g–treated THP-1 cells (5 3 105/ml) were stimulated with medium alone, 2.5 mg/ml CL075, DOTAP alone, or 2.5 mg/ml ssRNA40 complexed to DOTAP. After 12 h, total RNA was extracted, and quantitative PCR was performed using primers for the IL-12p40 and TLR8 genes. Expression of genes was normalized to b-actin mRNA expression. Knockdown efficiency is shown (right panel). Representative data from two independent experiments are shown (mean 6 SD).

Article Snippet: Monocyte-derived macrophages (5 3 105 /ml) were pretreated with DC1 (20 mM) or DMSO for 4 h and then were stimulated with CL075 (2.5 mg/ml), ssRNA40 complexed to DOTAP (2.5 mg/ml), or LPS (1 mg/ml) or left untreated in the presence of inhibitors for another 24 h. To examine stepwise processing of TLR8-N, monocytes were treated with 10 mM z-FA-FMK in the presence of 20 ng/ml recombinant hGM-CSF for 24 or 48 h and then stimulated with indicated ligands for 24 h. IL-12p40 in culture supernatants was measured by ELISA (R&D Systems).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, SDS Page, Western Blot, Control, Real-time Polymerase Chain Reaction, Knockdown

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Endosomal localization of TLR8 confers distinctive proteolytic processing on human myeloid cells.

doi: 10.4049/jimmunol.1401375

Figure Lengend Snippet: FIGURE 5. Furin-like proprotein convertases and cathepsins are involved in stepwise processing of hTLR8. (A) Monocytes were treated with GM-CSF in the presence or absence of 10 mM z-FA-FMK for up to 3 d. At day 1, day 2, and day 3, cells were lysed, and lysates were subjected to SDS-PAGE under reducing conditions, followed by Western blotting with anti–TLR8-N mAb (upper panels). The day-1 and day-2 monocytes were stimulated with medium alone, CL075 (2.5 mg/ml), or ssRNA40 complexed to DOTAP (2.5 mg/ml) for 24 h. IL-12p40 in the culture supernatants was measured using ELISA (lower panel). (B) Monocyte-derived macrophages were pretreated with 20 mM DC1 for 4 h and then stimulated with medium alone, CL075 (2.5 mg/ml), ssRNA40 complexed to DOTAP (2.5 mg/ml), or LPS (1 mg/ml) for 24 h. IL-12p40 in the culture supernatants was measured using ELISA (left panel). Lysates of DC1-treated macrophages were analyzed by Western blotting with anti–TLR8-N mAb and anti–tubulin-a mAb (right panel). (C) The potential furin-like proprotein convertase–recognition sites in LRR14 and insertion loop of hTLR8 (upper panel). Furin-like proprotein covertase–recognition site is R/K-Xn- R/K (X, any amino acid residue; n = 0, 2, 4, or 6). HEK293 cells were transfected with empty vector, wild-type TLR8 or R467A/R470A/R472A/R473A TLR8 mutant plasmid together with NF-kB–luciferase reporter plasmid and phRL-TK (middle panel). Cells were stimulated with 2.5 mg/ml CL075 or were left untreated. Luciferase activity was measured 12 h after stimulation and expressed as fold induction relative to the activity of unstimulated cells (mean 6 SD). Cell lysates prepared in the reporter assay (medium stimulation) were subjected to SDS-PAGE (7.5%), followed by Western blotting with anti–TLR8- N mAb and anti–tubulin-a mAb (bottom panel). Closed arrowhead indicates full-length TLR8. Open arrowheads indicate premature and mature TLR8-N. Asterisk indicates high molecular mass band of TLR8. Representative data from two independent experiments are shown.

Article Snippet: Monocyte-derived macrophages (5 3 105 /ml) were pretreated with DC1 (20 mM) or DMSO for 4 h and then were stimulated with CL075 (2.5 mg/ml), ssRNA40 complexed to DOTAP (2.5 mg/ml), or LPS (1 mg/ml) or left untreated in the presence of inhibitors for another 24 h. To examine stepwise processing of TLR8-N, monocytes were treated with 10 mM z-FA-FMK in the presence of 20 ng/ml recombinant hGM-CSF for 24 or 48 h and then stimulated with indicated ligands for 24 h. IL-12p40 in culture supernatants was measured by ELISA (R&D Systems).

Techniques: SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay, Derivative Assay, Residue, Transfection, Plasmid Preparation, Mutagenesis, Luciferase, Activity Assay, Reporter Assay