Journal: Frontiers in Immunology

Article Title: Lack of Association Between Sex Hormones, MDSCs, LDGs and pDCs in Males and Females With Systemic Lupus Erythematosus

doi: 10.3389/fimmu.2022.888501

Figure Lengend Snippet: IRF7 expression is upregulated in SLE, independent of pDC expression. RNA was isolated from paxgene blood samples and RT-PCR was run using Taqman probes for signaling molecules within the TLR activation pathway: (A) IRF5 , (B) IRF7 , and (C) IRAK4 . A single HC male sample was set as the standard level of expression and all samples were quantified as the relative gene expression to this control. Each sample was then normalized to its %pDCs as determined by flow cytometry analysis as presented in Figure 1B . Comparisons were made using Kruskal-Wallis Test with Dunn’s test for multiple comparisons. Bars represent median and interquartile range. aSLE F (N = 15), iSLE F (N = 50), HC F (N = 14), aSLE M (N = 5), iSLE M (N = 6), HC M (N = 5). *p < 0.05; **p < 0.01.

Article Snippet: The following TaqmanTM primer sets (ThermoFisher Scientific, Waltham, MA) were used: IFI27 (Hs01086370_m1), IFI44L (Hs00199115_m1), IFIT1 (Hs00356631_g1), ISG15 (Hs00192713_m1), RSAD2 (Hs01057264_m1), SIGLEC1 (Hs00988063_m1), IRAK4 (Hs00211610_m1), IRF5 (Hs00158114_m1), IRF7 (Hs01014809_g1), 18S (Hs999999001_s1).

Techniques: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Activation Assay, Gene Expression, Control, Flow Cytometry

Journal: Molecules

Article Title: A Novel IRAK4 Inhibitor DW18134 Ameliorates Peritonitis and Inflammatory Bowel Disease

doi: 10.3390/molecules29081803

Figure Lengend Snippet: Chemistry structure of compound DW18134 and its effects on the LPS-induced IRAK4 signaling transduction and secretion of cytokines in macrophages. ( A ) Chemical structure of DW18134. ( B ) Representative immunoblots of p-IRAK4 and p-IKK in PCM cells that were stimulated with LPS (5 µg/mL) and treated with or without indicated concentrations of DW18134 for 2 h. ( C ) Quantification of p-IRAK4 and p-IKK levels in PCM cells. ( D ) Representative immunoblots of p-IRAK4 and p-IKK in RAW264.7 cells that were stimulated with LPS and treated with or without DW18134. ( E ) p-IRAK4 and p-IKK levels in RAW264.7 cells were quantified. ( F , G ) Secretion levels of TNF-a and IL-6 in the supernatants of PCM and RAW264.7 cells were measured by ELISA. ( H , I ) Following the treatment of DW18134 for 24 h, cell viability of PCM and RAW 264.7 cells was determined by CCK-8 assays. A representative of three independent repeats is shown in ( B , D ). Data represent the mean ± standard deviation (SD) from three independent biological replicates in ( C , E – I ). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. LPS; ### p < 0.001 vs. control. ns: no significance.

Article Snippet: The antibody for Phospho-IRAK4 (Thr345/Ser346) (D6D7) Rabbit mAb(dilution, 1:1000; #11927), IRAK4 antibody (dilution, 1:1000; #4363), Phospho-IKKα/β (Ser176/180) (16A6) Rabbit mAb(dilution, 1:1000; #2697), NF-κB p65 (D14E12) Rabbit mAb(dilution, 1:1000; #8242), Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb(dilution, 1:1000; #3033), and actin (dilution, 1:20,000; # 60008-1-Ig, Proteintech, Cranbury, NJ, USA) were purchased from Cell Signaling Technology (CST, Boston, MA, USA).

Techniques: Transduction, Western Blot, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Standard Deviation, Control

Journal: Molecules

Article Title: A Novel IRAK4 Inhibitor DW18134 Ameliorates Peritonitis and Inflammatory Bowel Disease

doi: 10.3390/molecules29081803

Figure Lengend Snippet: The IC 50 values of compounds against IRAK4.

Article Snippet: The antibody for Phospho-IRAK4 (Thr345/Ser346) (D6D7) Rabbit mAb(dilution, 1:1000; #11927), IRAK4 antibody (dilution, 1:1000; #4363), Phospho-IKKα/β (Ser176/180) (16A6) Rabbit mAb(dilution, 1:1000; #2697), NF-κB p65 (D14E12) Rabbit mAb(dilution, 1:1000; #8242), Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb(dilution, 1:1000; #3033), and actin (dilution, 1:20,000; # 60008-1-Ig, Proteintech, Cranbury, NJ, USA) were purchased from Cell Signaling Technology (CST, Boston, MA, USA).

Techniques:

Journal: Molecules

Article Title: A Novel IRAK4 Inhibitor DW18134 Ameliorates Peritonitis and Inflammatory Bowel Disease

doi: 10.3390/molecules29081803

Figure Lengend Snippet: DW18134 reduced macrophage infiltration and pro-inflammatory gene expression and abrogated IRAK4 signaling pathway activation in LPS-induced peritonitis mice ( n = 8). Immunohistochemical staining of macrophage infiltration in the liver of mice with peritonitis. Includes quantitative data ( A ) and representative images ( B ). ( C ) Tnfa , Ill6 , and Il1b gene expression from liver homogenates measured by RT-qPCR. ( D ) Expression of p-IRAK4, p-IKK, and p-p65 in liver tissue of peritonitis mice treated with DW18134 or PF-06650833. ( E ) Quantification of p-IRAK4, p-IKK, and p-p65 levels in liver tissue based on three independent biological replicates. Data are expressed as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. LPS; ### p < 0.001 vs. control. ns: no significance.

Article Snippet: The antibody for Phospho-IRAK4 (Thr345/Ser346) (D6D7) Rabbit mAb(dilution, 1:1000; #11927), IRAK4 antibody (dilution, 1:1000; #4363), Phospho-IKKα/β (Ser176/180) (16A6) Rabbit mAb(dilution, 1:1000; #2697), NF-κB p65 (D14E12) Rabbit mAb(dilution, 1:1000; #8242), Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb(dilution, 1:1000; #3033), and actin (dilution, 1:20,000; # 60008-1-Ig, Proteintech, Cranbury, NJ, USA) were purchased from Cell Signaling Technology (CST, Boston, MA, USA).

Techniques: Gene Expression, Activation Assay, Immunohistochemical staining, Staining, Quantitative RT-PCR, Expressing, Control

Journal: Molecules

Article Title: A Novel IRAK4 Inhibitor DW18134 Ameliorates Peritonitis and Inflammatory Bowel Disease

doi: 10.3390/molecules29081803

Figure Lengend Snippet: DW18134 reduced macrophage infiltration and the expression of inflammatory factors and restored the intestinal barrier in mice with DSS ( n = 6). ( A , B ) Immunohistochemistry was used to detect macrophage infiltration in the colorectum. ( C , D ) Gene expression levels of tight junction proteins and Mucin2 were detected in the colon of IBD mice by RT-qPCR. ( E ) The mRNA expression of cytokines Tnfa , Il6 , and Il1b in colonic tissues. ( F ) Representative immunoblots of p-IRAK4 and p-IKK in the colonic homogenates from each group. p-IRAK4 and p-IKK levels were quantified (right panel). Quantified data represent the mean ± SD from three independent biological replicates. * p < 0.05, ** p < 0.01 vs. 3.5%DSS; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. control. ns: no significance.

Article Snippet: The antibody for Phospho-IRAK4 (Thr345/Ser346) (D6D7) Rabbit mAb(dilution, 1:1000; #11927), IRAK4 antibody (dilution, 1:1000; #4363), Phospho-IKKα/β (Ser176/180) (16A6) Rabbit mAb(dilution, 1:1000; #2697), NF-κB p65 (D14E12) Rabbit mAb(dilution, 1:1000; #8242), Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb(dilution, 1:1000; #3033), and actin (dilution, 1:20,000; # 60008-1-Ig, Proteintech, Cranbury, NJ, USA) were purchased from Cell Signaling Technology (CST, Boston, MA, USA).

Techniques: Expressing, Immunohistochemistry, Gene Expression, Quantitative RT-PCR, Western Blot, Control