Journal: Scientific Reports

Article Title: Development of a cell system for siRNA screening of pathogen responses in human and mouse macrophages

doi: 10.1038/srep09559

Figure Lengend Snippet: (a + b) Dose response of (a) cytosol-to-nuclear translocation of the GFP-relA fusion at 30 min (120 min for CpG) and (b) tnf promoter-driven mCherry expression at 12 hr after treatment of RAW G9 cells with LPS (0, 0.5, 1, 5, 12.5 and 25 ng/ml), P3C (0, 62.5, 125, 250, 500 and 1000 nM), P2C (0, 7.8, 31.25, 62.5, 125 and 250 nM), PGN (1, 15, 37.5, 75, 150 and 300 ng/ml), FLG (0, 0.1, 1, 10, 100 and 1000 ng/ml), R848 (0, 0.37, 0.75, 1.5, 3 and 6 μM), CpG (0, 0.35, 0.7, 1.4, 2.8 and 5.6 μg/ml) or pI:C (0, 12.5, 25 and 50 μg/ml). (c) Dose response of the TNF firefly/ UBC renilla luciferase expression ratio in THP1 B5 cells at 4 hr after treatment with LPS (0, 0.1, 1, 10 and 100 ng/ml), P3C (0, 1, 10, 100 and 1000 nM), P2C (0, 0.1, 1, 10 and 100 nM), PGN (0, 1, 10 and 100 μg/ml), FLG (0, 1, 10, 100 and 1000 ng/ml), R848 (0, 0.1, 1, 10 and 50 μg/ml), CpG (0, 1, 10, 100 and 1000 nM) or pI:C (0, 1, 10 and 100 μg/ml). Data are representative of three experiments (a–c; mean + s.d.). **P < 0.01, ***P < 0.001, ****P < 0.0001 (two-tailed t test).

Article Snippet: TLR ligand sources: LPS was from Alexis Biochemicals, Salmonella minnesota R595 TLRgrade, ALX-581-008-L002; Pam3CSK4 (P3C) was from EMC Microcollections, cat# L2000; PGN was from Sigma, peptidoglycan from Staphylococcus aureus, Cat.# 77140; R848 was from InvivoGen, tlrl-r848; Pam2CSK4 (P2C) was from EMC Microcollections, cat# L2020; Flagellin was from Invivogen, FLA-ST ultrapure, tlrl-epstfla; Lipid A was from Avanti Polar Lipids, 699500P; CpG was from IDT; poly I:C was from Invivogen, tlrl-picw.

Techniques: Translocation Assay, Expressing, Luciferase, Two Tailed Test

Journal: Scientific Reports

Article Title: Development of a cell system for siRNA screening of pathogen responses in human and mouse macrophages

doi: 10.1038/srep09559

Figure Lengend Snippet: (a–d) Time course of (a + b) cytosol-to-nuclear translocation of the GFP-relA fusion and (c + d) Tnf promoter-driven mCherry expression in RAW G9 cells after treatment with (a + c) 10 ng/ml LPS, 250 nM P3C, 125 nM P2C and 3 μM R848 and (b + d) 100 nM CpG and 3 μM R848. (e) Time course of the TNF firefly/ UBC renilla luciferase expression ratio in THP1 B5 cells after treatment with 10 ng/ml LPS, 100 nM P3C, 10 nM P2C, 10 μg/ml PGN and 10 μg/ml R848. Data are representative of three experiments (a–e; mean + s.d.)

Article Snippet: TLR ligand sources: LPS was from Alexis Biochemicals, Salmonella minnesota R595 TLRgrade, ALX-581-008-L002; Pam3CSK4 (P3C) was from EMC Microcollections, cat# L2000; PGN was from Sigma, peptidoglycan from Staphylococcus aureus, Cat.# 77140; R848 was from InvivoGen, tlrl-r848; Pam2CSK4 (P2C) was from EMC Microcollections, cat# L2020; Flagellin was from Invivogen, FLA-ST ultrapure, tlrl-epstfla; Lipid A was from Avanti Polar Lipids, 699500P; CpG was from IDT; poly I:C was from Invivogen, tlrl-picw.

Techniques: Translocation Assay, Expressing, Luciferase

Journal: Scientific Reports

Article Title: Development of a cell system for siRNA screening of pathogen responses in human and mouse macrophages

doi: 10.1038/srep09559

Figure Lengend Snippet: (a) Human TNF-α reporter responses in THP1 B5 cells transfected with either non-targeting control or TLR pathway gene-specific siRNAs and treated for 4 hr with either 10 ng/ml LPS, 10 μg/ml R848 or 10 ng/ml FLG. (b) tnf promoter-driven mCherry expression at 16 hr and (c) Cytosol-to-nuclear translocation of the GFP-relA fusion at 40 min in RAWG9 cells transfected with either non-targeting control or TLR pathway gene-specific siRNAs and treated with 10 ng/ml LPS or 3 μM R848. 6 individual siRNAs per gene were used and the average reporter response calculated. Data are representative of two experiments and reporter responses are normalized to the levels observed with non-targeting control siRNA (a–c; mean + s.d.). **P < 0.01, ***P < 0.001, ****P < 0.0001 (two-tailed t test).

Article Snippet: TLR ligand sources: LPS was from Alexis Biochemicals, Salmonella minnesota R595 TLRgrade, ALX-581-008-L002; Pam3CSK4 (P3C) was from EMC Microcollections, cat# L2000; PGN was from Sigma, peptidoglycan from Staphylococcus aureus, Cat.# 77140; R848 was from InvivoGen, tlrl-r848; Pam2CSK4 (P2C) was from EMC Microcollections, cat# L2020; Flagellin was from Invivogen, FLA-ST ultrapure, tlrl-epstfla; Lipid A was from Avanti Polar Lipids, 699500P; CpG was from IDT; poly I:C was from Invivogen, tlrl-picw.

Techniques: Transfection, Expressing, Translocation Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Epigenetic changes in autoimmune monocytes contribute to disease and can be targeted by JAK inhibition

doi: 10.1101/2021.11.20.469222

Figure Lengend Snippet: ( A ) Principal component analysis of H3K27ac signal in HC PB, JIA PB and JIA SF monocytes. ( B and C ) MA plot of H3K27ac regions different between JIA SF and HC PB (left) and between JIA SF and JIA PB monocytes. Red dot indicate H3K27ac regions with a FDR<0.1. ( D ) Top 10 biological processes associated with genes that are significantly increased in JIA SF vs. JIA PB monocytes. ( E ) Selected genes associated with increased H3K27ac signal in JIA SF vs. JIA PB monocytes. ( F ) Gene set enrichment analysis of genes associated with an increased H3K27ac signal in JIA SF monocytes and genes differentially expressed within JIA SF monocytes. ( G ) Boxplot with 5%-95% whiskers displaying ΔRPKM (log2) values of genes associated with an increased H3K27ac signal in HC and JIA monocytes. ( H ) Selected genes associated with an increased H3K27ac signal and increased in JIA. ( I ) Gene set enrichment analysis of genes decreased with JQ1 and genes differentially expressed within JIA SF monocytes. ( J ) Selected genes decreased with JQ1. ( K ) Boxplot with 5%-95% whiskers displaying ΔRPKM (log2) values of genes increased in JIA in JIA samples treated with or without JQ1. P values for G and K were calculated using a Wilcoxon-matched pairs signed rank test. * = p<0.05; ** = p<0.01, *** = p<0.001.

Article Snippet: Subsequently, CD14 + cells were cultured o/n with 100 ng/mL LPS (Invivogen) and treated with 300 nM JQ1(-) or JQ1(+) (ApexBio) in RPMI 1640 + GlutaMAX (Life Technologies) supplemented with 100 U/ml penicillin (Gibco), 100 mg/ml streptomycin (Gibco) and 10% heat-inactivated human AB-positive serum (Invitrogen) at 37°C in 5% CO 2 .

Techniques: