Journal: Oncotarget

Article Title: Middle east respiratory syndrome corona virus spike glycoprotein suppresses macrophage responses via DPP4-mediated induction of IRAK-M and PPARγ

doi: 10.18632/oncotarget.14754

Figure Lengend Snippet: A ., B . THP-1 macrophages were infected with lentiviral particles pseudotyped with MERS wild type (wt) or the D510A mutant S glycoprotein (D510A) of MERS-CoV, or left uninfected (mock) in the presence or absence of the DPP4 inhibitor sitagliptin or siRNA targeting DPP4. 24 hours post-infection RNA was isolated and the levels of IRAK-M A ., B . and PPARγ C ., D . and IL-10 E ., F . were measured. ns: not significant, * p < 0.05, ** p < 0.01

Article Snippet: Cells were cultured for 24 hours in the presence of the transfection reagents prior to infection and LPS treatment, the following, pre-validated siRNAs used were: IRAK-M siRNA (sc-39098, Santa-Cruz), PPARγ siRNA (sc-29455, Santa-Cruz), CD26 siRNA (sc-42762, Santa-Cruz) and Silencer Negative Control (SCR) siRNA (AM4611g, Ambion).

Techniques: Infection, Mutagenesis, Isolation

Journal: Inflammation

Article Title: CircIRAK3 Promotes Neutrophil Extracellular Trap Formation by Improving the Stability of ELANE mRNA in Sepsis

doi: 10.1007/s10753-024-02206-z

Figure Lengend Snippet: Identification and characterization of circIRAK3 in neutrophils of septic patients. A Volcano plot of differentially expressed circRNAs in neutrophils of septic patients relative to healthy volunteers. B Heatmap for top 12 up-regulated circRNAs in neutrophils from patients with sepsis. C PCR amplification using divergent or convergent primers against complementary DNA (cDNA) or genomic DNA (gDNA) from human neutrophils. D RT-qPCR to check for the expression of up-regulated circRNAs in neutrophils of sepsis patients ( n = 10) and healthy donors ( n = 10). Data shown are mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.0001, **** P < 0.0001. E Schematic diagram illustrating the genomic location and back-splicing of circIRAK3. F The back-splicing junction site of circIRAK3 was confirmed by PCR amplification and Sanger sequencing. G RNase R treatment followed by RT-qPCR of circIRAK3 and it’s host gene IRAK3. GAPDH represents mRNA. **** P < 0.0001, ns, no significance. H Actinomycin D (Act D) treatment of neutrophils followed by RT-qPCR. Linear mRNA controls: GAPDH and IRAK3. **** P < 0.0001, ns, no significance. I Cytoplasm/nucleus fraction isolation of neutrophils followed by RT-qPCR. GAPDH represents cytoplasmic RNA and MALAT1 represents nuclear RNA. J Images of FISH for circIRAK3. 18S, cytoplasmic RNA control; U6, nuclear RNA control

Article Snippet: Primary antibodies of IRAK3, MPO, PAD4, ELAVL1 and GAPDH and goat anti-mouse IgG-HRP antibody were from Proteintech, China.

Techniques: Amplification, Quantitative RT-PCR, Expressing, Sequencing, Isolation, Control

Journal: Inflammation

Article Title: CircIRAK3 Promotes Neutrophil Extracellular Trap Formation by Improving the Stability of ELANE mRNA in Sepsis

doi: 10.1007/s10753-024-02206-z

Figure Lengend Snippet: CircIRAK3 contributed to NET formation in dHL-60 neutrophil-like cells. A The expression level of circIRAK3 and IRAK3 mRNA in shcircIRAK3- transfected HL-60 cells, **** P < 0.0001, ns, no significance. B Western blot analysis of IRAK3 in circIRAK3- knockdown HL-60 cells, ns, no significance. C Reduced NET formation in shcircIRAK3- transfected dHL-60 stimulated with 100 nM PMA for 4 h. Left: Representative immunofluorescence images of NETs. The cells stained with DAPI (blue), MPO (green) and Cit-H3 (red). Right: Quantification of NETs release from dHL-60 cells. The percentage of NETs was calculated as the average of ten fields normalized to the total number of dHL-60 cells, *** P < 0.001, **** P < 0.0001. D The expression level of circIRAK3 and IRAK3 mRNA in circIRAK3- overexpressed dHL-60 cells, **** P < 0.0001, ns, no significance. E Western blot analysis of IRAK3 in circIRAK3-overexpressed dHL-60 cells, ns, no significance. F Increased NET formation in circIRAK3-overexpressed dHL-60 stimulated with 100 nM PMA for 4 h. Left: Representative immunofluorescence images of NETs. The cells stained with DAPI (blue), MPO (green) and Cit-H3 (red). Right: Quantification of NETs release from dHL-60 cells, * P < 0.05

Article Snippet: Primary antibodies of IRAK3, MPO, PAD4, ELAVL1 and GAPDH and goat anti-mouse IgG-HRP antibody were from Proteintech, China.

Techniques: Expressing, Transfection, Western Blot, Knockdown, Immunofluorescence, Staining

Journal: Inflammation

Article Title: CircIRAK3 Promotes Neutrophil Extracellular Trap Formation by Improving the Stability of ELANE mRNA in Sepsis

doi: 10.1007/s10753-024-02206-z

Figure Lengend Snippet: Schematic illustration of the mechanism whereby circIRAK3 regulates ELANE mRNA stability via ELAVL1. In nuclear, circIRAK3 is generated by backsplicing of the IRAK3 gene and exported to the cytoplasm. During sepsis, more circIRAK3 molecules are expressed to stabilize ELANE mRNA through their interactions with ELAVL1, thus facilitating chromatin decondensation and NET formation under inflammatory stimulation

Article Snippet: Primary antibodies of IRAK3, MPO, PAD4, ELAVL1 and GAPDH and goat anti-mouse IgG-HRP antibody were from Proteintech, China.

Techniques: Generated

Journal: PLoS ONE

Article Title: PPAR Agonist-Induced Reduction of Mcp1 in Atherosclerotic Plaques of Obese, Insulin-Resistant Mice Depends on Adiponectin-Induced Irak3 Expression

doi: 10.1371/journal.pone.0062253

Figure Lengend Snippet: Primers used for qRT-PCR analysis.

Article Snippet: To detect protein levels, membranes were incubated with primary antibodies against β-actin (Cell Signaling Technology) and Irak3 (Rockland Immunochemicals).

Techniques:

Journal: PLoS ONE

Article Title: PPAR Agonist-Induced Reduction of Mcp1 in Atherosclerotic Plaques of Obese, Insulin-Resistant Mice Depends on Adiponectin-Induced Irak3 Expression

doi: 10.1371/journal.pone.0062253

Figure Lengend Snippet: ( A ) Representative Mac-3 staining of aortic sinus plaques of placebo-, fenofibrate- and rosiglitazone-treated DKO mice at 24 weeks. ( B ) Gene expression in the aorta was analyzed by measuring relative RNA levels using qRT-PCR for Tnfα , IL6 , Mcp1 and Irak3 . Data are means ± SEM. Scale bar = 500 µm. * P <0.05, ** P <0.01 and *** P <0.001 DKO compared with C57BL/6 J mice; $$ P <0.01 and $$$ P <0.001 PPAR agonist-treated compared with placebo-treated DKO mice; £ P <0.05, ££ P <0.01 and £££ P <0.001 rosiglitazone-treated compared with fenofibrate-treated DKO mice.

Article Snippet: To detect protein levels, membranes were incubated with primary antibodies against β-actin (Cell Signaling Technology) and Irak3 (Rockland Immunochemicals).

Techniques: Staining, Gene Expression, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: PPAR Agonist-Induced Reduction of Mcp1 in Atherosclerotic Plaques of Obese, Insulin-Resistant Mice Depends on Adiponectin-Induced Irak3 Expression

doi: 10.1371/journal.pone.0062253

Figure Lengend Snippet: ( A ) Soluble Mcp1 protein levels in DKO BMDM exposed to 50 µM fenofibrate, 10 µM rosiglitazone or 5 µM GW9662 for 24 hours as determined by ELISA. Data are means ± SEM; n = 16 from three different mice. $$ P <0.01 compared with fenofibrate-treated BMDM. ( B ) Irak3 RNA and protein levels of DKO BMDM exposed to 1 or 10 µg/mL globular adiponectin for 24 hours as determined by qRT-PCR and Western blotting. Data are means ± SEM; n = 6. *** P <0.001 compared with DKO BMDM; $ P <0.05 and $$ P <0.01 compared with DKO BMDM exposed to 1 µg/mL globular adiponectin. ( C ) Soluble Mcp1 protein levels (n = 18 from three different mice), NFκB p50 DNA binding activity (n = 8 from two different mice) and mROS production (n = 6) in IRAK3 −/− BMDM exposed to 50 µM fenofibrate or 10 µM rosiglitazone for 24 hours as determined by ELISA and flow cytometry. Data are means ± SEM. * P <0.05, ** P <0.01 and *** P <0.001 compared with C57BL/6 J BMDM; $ P <0.05 and $$$ P <0.001 compared with IRAK3 −/− BMDM; £££ P <0.001 compared with fenofibrate-treated BMDM. Abbreviations: BMDM, bone marrow-derived macrophages; mROS, mitochondrial reactive oxygen species.

Article Snippet: To detect protein levels, membranes were incubated with primary antibodies against β-actin (Cell Signaling Technology) and Irak3 (Rockland Immunochemicals).

Techniques: Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot, Binding Assay, Activity Assay, Flow Cytometry, Derivative Assay

Journal: PLoS ONE

Article Title: PPAR Agonist-Induced Reduction of Mcp1 in Atherosclerotic Plaques of Obese, Insulin-Resistant Mice Depends on Adiponectin-Induced Irak3 Expression

doi: 10.1371/journal.pone.0062253

Figure Lengend Snippet: Plaque volume was determined by measuring lipid (oil red O)-stained surfaces in subsequent sections; macrophages were stained with anti-Mac-3 antibody. Gene expression in the aorta was analyzed by measuring relative RNA levels using qRT-PCR for Pparγ , Mcp1, Irak3 and Adipoq . Data are means ± SEM. ** P <0.01 and *** P <0.001 HFD-fed compared with SD-fed LDL-receptor deficient mice. Abbreviations: HFD, high fat diet; SD, standard diet.

Article Snippet: To detect protein levels, membranes were incubated with primary antibodies against β-actin (Cell Signaling Technology) and Irak3 (Rockland Immunochemicals).

Techniques: Staining, Gene Expression, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: PPAR Agonist-Induced Reduction of Mcp1 in Atherosclerotic Plaques of Obese, Insulin-Resistant Mice Depends on Adiponectin-Induced Irak3 Expression

doi: 10.1371/journal.pone.0062253

Figure Lengend Snippet: The schematic draw demonstrates the anti-atherosclerotic properties of the PPARγ agonist rosiglitazone. Treatment with rosiglitazone improves the adipocyte function characterized by a decrease in adipocyte size, a reduction in adipose tissue macrophages and an increased expression of anti-inflammatory adiponectin. The increase in blood adiponectin and de novo adiponectin production in atherosclerotic lesions is necessary for the upregulation of Irak3 in plaque macrophages, which is crucial for the indirect rosiglitazone-mediated decrease in Mcp1 secretion. Abbreviations: Mφ, macrophages; ROS, reactive oxygen species.

Article Snippet: To detect protein levels, membranes were incubated with primary antibodies against β-actin (Cell Signaling Technology) and Irak3 (Rockland Immunochemicals).

Techniques: Expressing