Journal: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association

Article Title: SerpinB9 expression in human renal tubular epithelial cells is induced by triggering of the viral dsRNA sensors TLR3, MDA5 and RIG-I.

doi: 10.1093/ndt/gfr690

Figure Lengend Snippet: Fig. 1. TLR3 triggering induces serpinB9 expression in human TECs. Primary TECs were stimulated for 16 h (light gray bars) or 40 h (dark gray bars) with 100 ng/mL TNF-α, 100 ng/mL IFNγ, 100 ng/mL IL-1β, 1000 pg/mL IL-6, 100 μg/mL LTA (TLR2 ligand), 100 μg/mL poly(I:C) (pIC, TLR3 ligand), 10 μg/mL LPS (TLR4 ligand), 100 μg/mL imiquimod (ImiQ, TLR7 ligand) or 10 μg/mL CpG oligonucleotides (CpG, TLR9 ligand). For each stimulus, three concentrations with a one-log difference were tested, and the highest concentration is depicted in the figure. (A) SerpinB9 expression was analysed, by quantitative PCR. An increase of mRNA above 2-fold (dotted line) was considered significant. (B and C) Protein levels of serpinB9 were determined by western blot analysis. Representative blots of serpinB9 expression in TECs stimulated for 16 h are depicted below the graph. The bars represent the fold change in serpinB9 expression compared to unstimulated TECs (mean ± SEM, n ≥3). **P < 0.01. (C) The TLR3 inhibitor chloroquine (HCQ, 10–100 μM) was administered 30 min prior to stimulation with poly(I:C) (pIC, 0.1–500 μg/mL, 16 h).

Article Snippet: Proteins (50 μg per lane) were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane and probed with primary antibodies against serpinB9 (mouse IgG1, clone PI9-17; kindly provided by Prof. S.M. van Ham, Sanquin, Amsterdam, The Netherlands [11], or mouse IgG1, clone 7D8; AbD Serotec, Dusseldorf, Germany), serpinB8 (mouse IgG; Abcam, Cambridge, UK), TLR3 (mouse IgG1, clone 40C1285.6; Abnova GmbH, Heidelberg, Germany), MDA5 (goat Ig; Imgenex, San Diego, CA), RIG-I (mouse IgG1, clone Alme-1; Enzo Life Sciences, Zandhoven, Belgium) and actin (goat Ig, I19; Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Expressing, Concentration Assay, Real-time Polymerase Chain Reaction, Western Blot

Journal: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association

Article Title: SerpinB9 expression in human renal tubular epithelial cells is induced by triggering of the viral dsRNA sensors TLR3, MDA5 and RIG-I.

doi: 10.1093/ndt/gfr690

Figure Lengend Snippet: Fig. 3. The dsRNA receptors TLR3, MDA5 and RIG-I upregulate serpinB9 expression in TECs independent of new protein synthesis. Primary TECs were stimulated for 16 h with 0.01–10 μg/mL poly(I:C) (pIC) or 0.1 μg/mL 3pRNA. The transfection reagent Fugene HD (FG) was used to accomplish cytoplasmic delivery. SerpinB9 expression was determined by western blot analysis (A) or quantitative PCR (B). The bars represent the fold change in serpinB9 expression compared to unstimulated cells (mean ± SEM, n = 3). (B) The inhibitors Bay 11-7082 (Bay, 5 μM) or cycloheximide (CHX, 5 μg/mL) were administered 30 min prior to dsRNA receptor activation; *P < 0.05.

Article Snippet: Proteins (50 μg per lane) were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane and probed with primary antibodies against serpinB9 (mouse IgG1, clone PI9-17; kindly provided by Prof. S.M. van Ham, Sanquin, Amsterdam, The Netherlands [11], or mouse IgG1, clone 7D8; AbD Serotec, Dusseldorf, Germany), serpinB8 (mouse IgG; Abcam, Cambridge, UK), TLR3 (mouse IgG1, clone 40C1285.6; Abnova GmbH, Heidelberg, Germany), MDA5 (goat Ig; Imgenex, San Diego, CA), RIG-I (mouse IgG1, clone Alme-1; Enzo Life Sciences, Zandhoven, Belgium) and actin (goat Ig, I19; Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Expressing, Transfection, Western Blot, Real-time Polymerase Chain Reaction, Activation Assay

Journal: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association

Article Title: SerpinB9 expression in human renal tubular epithelial cells is induced by triggering of the viral dsRNA sensors TLR3, MDA5 and RIG-I.

doi: 10.1093/ndt/gfr690

Figure Lengend Snippet: Fig. 4. SerpinB9 expression in the kidney is enhanced during viral infection. (A) SerpinB9 transcription in frozen biopsy specimens obtained from stable patients (n = 5) and patients infected with CMV (n = 6), EBV (n = 4) or BKV (n = 7) depicted as relative expression (median). *P < 0.05, **P < 0.01. (B and C) Immunohostochemical stainings for serpinB9 on formalin-fixed paraffin-embedded tissue slides. (B) Semi-quantitative scoring of the tubular SerpinB9 expression, scored as described in the Materials and Methods (median). (C) Representative pictures of the serpinB9 staining pattern observed in stable, CMV, EBV and BKV biopsies, with serpinB9 expression found in the tubuli (black arrows) and infiltrating lymphocytes (red arrow) (magnification 200 × ).

Article Snippet: Proteins (50 μg per lane) were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane and probed with primary antibodies against serpinB9 (mouse IgG1, clone PI9-17; kindly provided by Prof. S.M. van Ham, Sanquin, Amsterdam, The Netherlands [11], or mouse IgG1, clone 7D8; AbD Serotec, Dusseldorf, Germany), serpinB8 (mouse IgG; Abcam, Cambridge, UK), TLR3 (mouse IgG1, clone 40C1285.6; Abnova GmbH, Heidelberg, Germany), MDA5 (goat Ig; Imgenex, San Diego, CA), RIG-I (mouse IgG1, clone Alme-1; Enzo Life Sciences, Zandhoven, Belgium) and actin (goat Ig, I19; Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Expressing, Infection, Staining

Journal: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association

Article Title: SerpinB9 expression in human renal tubular epithelial cells is induced by triggering of the viral dsRNA sensors TLR3, MDA5 and RIG-I.

doi: 10.1093/ndt/gfr690

Figure Lengend Snippet: Fig. 5. Schematic overview of dsRNA receptor signalling in TECs. Human TECs express the dsRNA sensors TLR3, MDA5 and RIG-I. TLR3 is located in the membrane of endolysosomes and becomes activated after endocytosis of viral particles or dsRNA. The cytoplasmic receptors MDA5 and RIG-I recognize dsRNA generated during viral replication in infected cells. The three dsRNA receptors activate the same transcription factors; namely NF-κB and IRF-3/7. Triggering of NF-κB induces transcription of proinflammatory cytokines like TNF-α, while IRF-3/7 initiate gene expression of Type I IFNs. Transcription of serpinB9 and the dsRNA receptors themselves is regulated by both NF- κB and IRFs. In addition, Type I IFNs trigger the IFN receptor leading to IRF-9-mediated transcription of the dsRNA receptor genes. This feed forward loop renders TECs more sensitive to dsRNA.

Article Snippet: Proteins (50 μg per lane) were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane and probed with primary antibodies against serpinB9 (mouse IgG1, clone PI9-17; kindly provided by Prof. S.M. van Ham, Sanquin, Amsterdam, The Netherlands [11], or mouse IgG1, clone 7D8; AbD Serotec, Dusseldorf, Germany), serpinB8 (mouse IgG; Abcam, Cambridge, UK), TLR3 (mouse IgG1, clone 40C1285.6; Abnova GmbH, Heidelberg, Germany), MDA5 (goat Ig; Imgenex, San Diego, CA), RIG-I (mouse IgG1, clone Alme-1; Enzo Life Sciences, Zandhoven, Belgium) and actin (goat Ig, I19; Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Membrane, Generated, Infection, Gene Expression

Journal: International Journal of Molecular Sciences

Article Title: The Potential Role of DNA Methylation in Abdominal Aortic Aneurysms

doi: 10.3390/ijms160511259

Figure Lengend Snippet: Outline of the study. We collected blood samples and isolated mononuclear cells for DNA and RNA extraction from four all male groups: AAA smokers, AAA non-smokers, control smokers and control non-smokers. Methylation data were obtained using the Illumina 450k HumanMethylation Bead Chip and analyzed using the R language and multiple Bioconductor packages. Principal component analysis and linear analysis of CpG island subsets identified four regions with significant differences in methylation with respect to AAA: kelch-like family member 35 ( KLHL35 ), calponin 2 ( CNN2 ), serpin peptidase inhibitor clade B (ovalbumin) member 9 ( SERPINB9 ), and adenylate cyclase 10 pseudogene 1 ( ADCY10P1 ) (pseudogene). Follow-up studies included RT-PCR and immunostaining for CNN2 and SERPINB9 . Symbols: AAA, abdominal aortic aneurysm; FFPE, formalin-fixed paraffin-embedded tissue; QC, quality control.

Article Snippet: SERPINB9 , 5272 , CAP3, PI9, SERPINB9 , Serpin peptidase inhibitor, clade B (ovalbumin), member 9 , TA312970 , OriGene, Rockville, MD, USA , Rabbit polyclonal , 1:4667.

Techniques: Isolation, RNA Extraction, Control, Methylation, Reverse Transcription Polymerase Chain Reaction, Immunostaining, Formalin-fixed Paraffin-Embedded

Journal: International Journal of Molecular Sciences

Article Title: The Potential Role of DNA Methylation in Abdominal Aortic Aneurysms

doi: 10.3390/ijms160511259

Figure Lengend Snippet: CpGIs in the subset analyzed using ordinary least squares (OLS) linear regression models.

Article Snippet: SERPINB9 , 5272 , CAP3, PI9, SERPINB9 , Serpin peptidase inhibitor, clade B (ovalbumin), member 9 , TA312970 , OriGene, Rockville, MD, USA , Rabbit polyclonal , 1:4667.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: The Potential Role of DNA Methylation in Abdominal Aortic Aneurysms

doi: 10.3390/ijms160511259

Figure Lengend Snippet: Genes demonstrating differential DNA methylation with respect to AAA, age, and smoking.

Article Snippet: SERPINB9 , 5272 , CAP3, PI9, SERPINB9 , Serpin peptidase inhibitor, clade B (ovalbumin), member 9 , TA312970 , OriGene, Rockville, MD, USA , Rabbit polyclonal , 1:4667.

Techniques: DNA Methylation Assay, Biomarker Discovery

Journal: International Journal of Molecular Sciences

Article Title: The Potential Role of DNA Methylation in Abdominal Aortic Aneurysms

doi: 10.3390/ijms160511259

Figure Lengend Snippet: Comparison of CNN2 and SERPINB9 mRNA levels in PBMC of AAA smokers ( n = 14), AAA non-smokers ( n = 12), control smokers ( n = 10) and control non-smokers ( n = 10). The C t difference was calculated by subtracting the C t value of the RPL gene (housekeeping gene) from the C t value of CNN2 (or SERPINB9 ) for each sample. The difference was then subtracted from the value 40 (highest cycle count) to obtain values in which larger value means higher expression and lower value means lower expression level. Each sample was run in triplicate. Box-and-whisker plots are presented, in which the thick horizontal bars in the boxes indicate median values, boxes indicate interquartile range, whiskers indicate range of non-outlier values, and open circles indicate outliers less than 3 interquartile range units. Gene symbols available from the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/ ) were used. See and for details on the donors.

Article Snippet: SERPINB9 , 5272 , CAP3, PI9, SERPINB9 , Serpin peptidase inhibitor, clade B (ovalbumin), member 9 , TA312970 , OriGene, Rockville, MD, USA , Rabbit polyclonal , 1:4667.

Techniques: Comparison, Control, Expressing, Whisker Assay

Journal: International Journal of Molecular Sciences

Article Title: The Potential Role of DNA Methylation in Abdominal Aortic Aneurysms

doi: 10.3390/ijms160511259

Figure Lengend Snippet: Primary antibodies used in immunohistological analyses.

Article Snippet: SERPINB9 , 5272 , CAP3, PI9, SERPINB9 , Serpin peptidase inhibitor, clade B (ovalbumin), member 9 , TA312970 , OriGene, Rockville, MD, USA , Rabbit polyclonal , 1:4667.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: The Potential Role of DNA Methylation in Abdominal Aortic Aneurysms

doi: 10.3390/ijms160511259

Figure Lengend Snippet: Immunohistochemical staining for CNN2 and SERPINB9 in human aneurysmal and non-aneurysmal abdominal aorta. See and for details on the aortic tissues and antibodies used, respectively. Scale bar = 100 μm.

Article Snippet: SERPINB9 , 5272 , CAP3, PI9, SERPINB9 , Serpin peptidase inhibitor, clade B (ovalbumin), member 9 , TA312970 , OriGene, Rockville, MD, USA , Rabbit polyclonal , 1:4667.

Techniques: Immunohistochemical staining, Staining

Journal: International Journal of Molecular Sciences

Article Title: The Potential Role of DNA Methylation in Abdominal Aortic Aneurysms

doi: 10.3390/ijms160511259

Figure Lengend Snippet: Network of interactions between CNN2 and SERPINB9 . Ingenuity Pathway Analysis ® (Qiagen’s Ingenuity Systems, Redwood City, CA, USA) tool was used for the analysis. Molecules are represented as nodes, and the biological relationship between two nodes is represented as a line. Solid lines represent direct interactions and dashed lines indirect interactions. All lines are supported by at least one literature citation or from canonical information stored in the Ingenuity Pathways Knowledge Base (Qiagen’s Ingenuity Systems). Nodes are displayed using various shapes that represent the functional class of the gene product.

Article Snippet: SERPINB9 , 5272 , CAP3, PI9, SERPINB9 , Serpin peptidase inhibitor, clade B (ovalbumin), member 9 , TA312970 , OriGene, Rockville, MD, USA , Rabbit polyclonal , 1:4667.

Techniques: Functional Assay