Journal: The Journal of Experimental Medicine

Article Title: Inherited IL-18BP deficiency in human fulminant viral hepatitis

doi: 10.1084/jem.20190669

Figure Lengend Snippet: Homozygous 40-nt deletion in IL18BP . (A) Pedigree of the family affected by FVH due to HAV. The patient is shown in black, whereas healthy individuals are shown in white. Where available, IL18BP mutation (NM_173042.2:c.508-19_528del) status is indicated in red. M, mutant. (B) Familial segregation of the mutation and its homozygous state in the patient were confirmed by Sanger sequencing. (C) Graph showing the predicted CADD scores and global AFs of the mutation found in the patient with FVH (red circle) and missense variants of IL18BP (blue circles) for which homozygotes were reported in GnomAD. The CADD-MSC score (90% confidence interval) for IL18BP is indicated by a dashed line. (D) The upper panel shows the exons (1–5) of the canonical IL18BP transcript; the bottom panel shows a diagram for IL-18BP. The signal peptide is highlighted in blue; the Ig domain is shown in red. Start and stop codons are indicated by an arrow and an asterisk, respectively. The c.508-19_528del is shown as a dashed box on the mRNA. The locations of IL18BP alleles from GnomAD are also shown on the protein diagram.

Article Snippet: Immunoblotting was performed with primary antibodies against the His-Tag (MA1-21315, 0.5 μg/ml; Thermo Fisher Scientific) and IL-18BP (AF119, 0.5 μg/ml; R&D Systems).

Techniques: Mutagenesis, Sequencing

Journal: The Journal of Experimental Medicine

Article Title: Inherited IL-18BP deficiency in human fulminant viral hepatitis

doi: 10.1084/jem.20190669

Figure Lengend Snippet: Impact of the IL18BP :c.508-19_528del on gene expression and function. (A) RT-qPCR showing IL18BP levels normalized against endogenous GAPDH expression in EBV-B cell lines from six healthy controls (black), the WT sibling (III.3, purple), and heterozygous family members: brother (III.2, red), father (II.4, blue), and mother (II.9, green). Relative IL18BP expression was determined by normalization against the mean value for WT cells, set to 1 (indicated by a dashed line). The values shown are the means of two independent experiments performed in duplicate. (B) Agarose gel electrophoresis showing aberrant splicing of the IL18BP mRNA in 3′ RACE on EBV-B cells from the heterozygous sibling (III.2), relative to a control cell line (C1) and the WT sibling (III.3). HPRT1 was used as the housekeeping gene control. (C) The nested PCR products from B were cloned, and colonies were sequenced. Diagram (left) and percentages (right) of WT (gray) and mutant (M1 in blue, M2 in red, and M3 in green) splice variants of the IL18BP transcript are shown. The start codon is located at position 1, and the stop codon is at 585, shown by an asterisk, on the WT transcript. The polyadenylation site is at position 1,252 and indicated by A n . (D) Expression levels of each splice variant (WT in gray, M1 in blue, M2 in red, and M3 in green) were determined and normalized against endogenous GAPDH expression levels by RT-qPCR on EBV-B cells from two healthy controls (C2 and C3) and family members. Graph shows the copy numbers of the mutant splice variants relative to the mean copy number for the WT allele in EBV-B cells from C2, C3, and III.3, which was set to 1 (indicated by a dashed line). The values are the means ± SEM of two independent experiments performed in duplicate. (E and F) Representative immunoblot images showing levels of the WT and mutant IL-18BP alleles, M1–M3 (E), and four missense alleles from GnomAD (F) in concentrated supernatants from transiently transfected COS7 cells. Immunoblotting was performed with the His tag antibody (top), and the membrane was then stripped and probed with the IL18BP antibody (bottom). (G) IL-18BP bioassay: IFN-γ production was measured in NK-92 cells stimulated with recombinant human IL-12 (100 pg/ml), IL-18 (10 ng/ml), and/or concentrated supernatants (100 µg/ml of total protein) of COS7 cells transiently transfected with either empty vector or the constructs expressing indicated IL-18BP variants. Graph is presented on a logarithmic scale with base of 10. The data are the means ± SEM of two independent experiments performed in duplicate using the supernatants shown in E and F and Fig. S1, F and G.

Article Snippet: Immunoblotting was performed with primary antibodies against the His-Tag (MA1-21315, 0.5 μg/ml; Thermo Fisher Scientific) and IL-18BP (AF119, 0.5 μg/ml; R&D Systems).

Techniques: Gene Expression, Quantitative RT-PCR, Expressing, Agarose Gel Electrophoresis, Control, Nested PCR, Clone Assay, Mutagenesis, Variant Assay, Western Blot, Transfection, Membrane, Bioassay, Recombinant, Plasmid Preparation, Construct

Journal: The Journal of Experimental Medicine

Article Title: Inherited IL-18BP deficiency in human fulminant viral hepatitis

doi: 10.1084/jem.20190669

Figure Lengend Snippet: Liver immunohistochemical profile of the patient. Liver tissue sections from a control individual, an unrelated patient with FVH due to HAV, and the deceased IL-18BP–deficient FVH patient reported in this study were subjected to immunohistochemical staining with the following markers: Hep Par-1, CD8, perforin, CD57, CD68, and IL-18. Representative zoom-in views of the original images at 400× magnification (Fig. S4) are shown. Hep Par-1 staining of IL-18BP–deficient patient’s liver tissue section displayed a background staining of macrophages, with lower intensity than hepatocytes. Some IL-18–positive hepatocytes and macrophages are indicated with blue and red arrows, respectively. Scale bar represents 50 µm.

Article Snippet: Immunoblotting was performed with primary antibodies against the His-Tag (MA1-21315, 0.5 μg/ml; Thermo Fisher Scientific) and IL-18BP (AF119, 0.5 μg/ml; R&D Systems).

Techniques: Immunohistochemical staining, Control, Staining

Journal: The Journal of Experimental Medicine

Article Title: Inherited IL-18BP deficiency in human fulminant viral hepatitis

doi: 10.1084/jem.20190669

Figure Lengend Snippet: IL-18/IL-18BP–mediated hepatotoxicity. (A and B) Coculture of mock- or HAV-infected hepatocytes (HepG2 and Huh7.5 cells) with NK-92 cells pretreated with IL-18, IL-18 + IL-18BP, or IL-18BP. HAV infection efficiencies in HepG2 and Huh7.5 cells were ∼40% and ~100%, respectively (Fig. S5 E; Materials and methods). The relative survival of calcein-AM–stained HepG2 or Huh7.5 cells was calculated based on the measurement of fluorescence retention within cells (A) and the amount of secreted albumin (B). Relative fluorescence and albumin levels were determined by normalization against the mean value for hepatocytes cocultured with NK92 cells without pretreatment (not treated [NT]), set to 100. A decrease in the fluorescence or in albumin levels indicates an increase in NK cell–induced hepatotoxicity. The data shown are the means ± SEM of three independent experiments performed in quadruplicate (n.s., not significant; **, P < 0.01; ***, P < 0.001; one-way ANOVA with Bonferroni correction for multiple comparisons). (C) A proposed model for IL-18BP deficiency underlying FVH. During the course of acute HAV infection in an otherwise healthy individual (left), IL-18 is secreted by macrophages in the liver. This cytokine activates lymphocytes, such as NK cells, inducing IFN-γ production and cytotoxicity to eliminate HAV-infected cells. IFN-γ also induces IL-18BP secretion by hepatocytes, macrophages, and other nonparenchymal cells (endothelial cells, fibroblasts, and hepatic stellate cells), to buffer IL-18 activity. However, in the absence of IL-18BP (right), excessive IL-18 activity leads to uncontrolled, massive immune-mediated hepatotoxicity and severe liver injury, as in the IL-18BP–deficient individual with FVH.

Article Snippet: Immunoblotting was performed with primary antibodies against the His-Tag (MA1-21315, 0.5 μg/ml; Thermo Fisher Scientific) and IL-18BP (AF119, 0.5 μg/ml; R&D Systems).

Techniques: Infection, Staining, Fluorescence, Activity Assay

Journal: Placenta

Article Title: Expression pattern and function of Notch2 in different subtypes of first trimester cytotrophoblast

doi: 10.1016/j.placenta.2015.01.009

Figure Lengend Snippet: Expression of Notch2 in first trimester placental and decidual tissues. Cytokeratin 7 (KRT7) and HLA-G were used to mark all CTB subtypes and EVTs. Nuclei are stained with DAPI. Representative examples showing localisation of Notch2 in cell column trophoblasts (first panel; dCCT, distal cell column trophoblast; pCCT, proximal cell column trophoblast; 12th week placenta, scale bars represent 100 μm), in interstitial cytotrophoblasts (iCTBs) of the decidua basalis (second panel; 12th week, scale bars represent 50 μm), and in intramural cytotrophoblasts (imCTB), associated with maternal blood vessels (fourth panel; 12th week decidua basalis, digitally zoomed). In all placentae analysed (n = 4, between 6th and 12th week of gestation) expression was weaker in pCCTs and villous cytotrophoblasts (vCTB) compared to non-proliferative, HLA-G + dCCTs. Inserts (i) depict digital magnifications, showing HLA-G and Notch2 in dCCTs. Notch2 was also present in the villous core (VC) as well as in decidual stromal cells (DSC), some of which showed nuclear staining as published . VE-cadherin (CDH5) stained endothelial cells (EC) in spiral arteries (SA) present in maternal decidua (third panel; 12th week decidua basalis, scale bars represent 100 μm). Partial disruption of the maternal EC layer/VE-cadherin staining depicted in (ii) suggested on-going vessel remodelling. imCTBs in (ii) and (iii) showed nuclear Notch2 staining. Selected areas (ii) and (iii) on the right-hand side represent magnified overlays of KRT7 + /Notch2 + imCTBs with nuclear N2ICD.

Article Snippet: For specific inhibition of Notch2 signalling, cultivated cells were treated with 0.4 μg/ml Notch2 blocking antibody or 0.4 μg/ml human IgG isotype control (Novus Biologicals, Littleton, CO).

Techniques: Expressing, Staining, Disruption

Journal: Placenta

Article Title: Expression pattern and function of Notch2 in different subtypes of first trimester cytotrophoblast

doi: 10.1016/j.placenta.2015.01.009

Figure Lengend Snippet: Expression of Notch2 and EVT markers in EGFR + and HLA-G + CTBs isolated from first trimester placental tissue. (A) Transcript levels of Notch2 , EGFR , HLA-G , integrin α1 ( ITGA1 ) and integrin α5 ( ITGA5 ) in the two different CTB populations using qRT-PCR. Mean values ± S.D. obtained from five different CTB isolations are shown. For relative quantification (AU, arbitrary units) values of each target gene were arbitrarily set to 1 in EGFR + CTBs. *p < 0.05. Consistent with EVT differentiation HLA-G , ITGA1 and ITGA5 mRNAs were elevated in HLA-G + CTBs compared to EGFR + CTBs, the latter expressing high levels of EGFR . (B) Protein levels of Notch2, EGFR, HLA-G and TCF-4 in the two purified CTB cell types analysed by Western blotting. In agreement with the mRNA expression data HLA-G + CTBs expressed increased levels of Notch2, HLA-G and TCF-4. The latter has been recently established as a marker of EVT . GAPDH was used as loading control.

Article Snippet: For specific inhibition of Notch2 signalling, cultivated cells were treated with 0.4 μg/ml Notch2 blocking antibody or 0.4 μg/ml human IgG isotype control (Novus Biologicals, Littleton, CO).

Techniques: Expressing, Isolation, Quantitative RT-PCR, Quantitative Proteomics, Purification, Western Blot, Marker, Control

Journal: Placenta

Article Title: Expression pattern and function of Notch2 in different subtypes of first trimester cytotrophoblast

doi: 10.1016/j.placenta.2015.01.009

Figure Lengend Snippet: siRNA-mediated Notch2 knockdown or antibody-mediated inhibition of Notch2-dependent signalling increase motility of primary CTBs and SGHPL-5 cells through fibronectin-coated transwells. (A) Migration in the presence of Notch2 siRNA (siN2) or non-targeting control (ntc). Bars represent mean values ± S.D. obtained from each three CTB isolations/SGHPL-5 cell experiments performed in duplicates. ntc was arbitrarily set to 100%. *p < 0.05. (B) Migration upon addition of Notch2 blocking antibody (N2AB) or IgG control (IgG ctrl). Bars represent mean values ± S.D. obtained from three experiments performed in duplicates. IgG control was arbitrarily set to 100%. *p < 0.05.

Article Snippet: For specific inhibition of Notch2 signalling, cultivated cells were treated with 0.4 μg/ml Notch2 blocking antibody or 0.4 μg/ml human IgG isotype control (Novus Biologicals, Littleton, CO).

Techniques: Knockdown, Inhibition, Migration, Control, Blocking Assay

Journal: Placenta

Article Title: Expression pattern and function of Notch2 in different subtypes of first trimester cytotrophoblast

doi: 10.1016/j.placenta.2015.01.009

Figure Lengend Snippet: Antibody-mediated inhibition of Notch2-dependent signalling in first trimester floating villous explant cultures and primary CTBs. Proliferation was analysed by EdU incorporation and counting of the EdU/DAPI ratio. (A) Representative pictures (scale bars represent 50 μm) showing immunofluorescent EdU staining in floating explant cultures treated with Notch2 blocking antibody (N2AB) or IgG control (IgG ctrl). Upper and lower panel show EdU labelling in HAI1 + vCTBs and KRT7 + CCTs. Nuclei are stained with DAPI. EdU + vCTBs and CCTs are indicated by arrows. CCT, cell column trophoblast; VC, villous core; vCTB, villous cytotrophoblasts. (B) Percentage of EdU + villous CTBs and CCTs. Each 14 explants isolated from three different placentae were evaluated in the presence of N2AB or IgG ctrl. Bar graphs represent mean values ± S.D. of 1200–1400 nuclei of vCTBs and 2400–2600 nuclei of CCTs per condition. (C) Percentage of EdU + primary CTBs after treatment with N2AB or IgG ctrl. Bar graphs represent mean values ± S.D. of three experiments performed in duplicates. n.s., not significant.

Article Snippet: For specific inhibition of Notch2 signalling, cultivated cells were treated with 0.4 μg/ml Notch2 blocking antibody or 0.4 μg/ml human IgG isotype control (Novus Biologicals, Littleton, CO).

Techniques: Inhibition, Staining, Blocking Assay, Control, Isolation

Journal: BMC Veterinary Research

Article Title: Notch2 signal is required for the maintenance of canine hemangiosarcoma cancer stem cell-like cells

doi: 10.1186/s12917-018-1624-8

Figure Lengend Snippet: 2 -ΔCt values of NOTCH2 and NOTCH4

Article Snippet: Membranes were either incubated with anti-human Notch2 intracellular domain antibody (R&D systems, MN, USA; 1:2000), anti-FLAG M2 monoclonal antibody (Sigma-Aldrich; 1:1000) or anti-Actin antibody clone C4 (Merck Millipore; 1:10,000) overnight at 4 °C.

Techniques:

Journal: BMC Veterinary Research

Article Title: Notch2 signal is required for the maintenance of canine hemangiosarcoma cancer stem cell-like cells

doi: 10.1186/s12917-018-1624-8

Figure Lengend Snippet: a Western blot analysis using anti-human Notch2 antibody. HeLa cell line was used as a positive control. b Notch2 protein expression levels in HSA cell lines cultured under the normal or serum-free condition, c Conceptual diagram of Notch2 protein and its mutants. d Gene expression levels of Notch target genes. Vec = cells transfected the empty vector. FL = cells overexpressing full length of Notch2. Ex = cells overexpressing dominant negative form of Notch2. In = cells overexpressing constitutive active form of Notch2. Gene expression levels of Vec were set to 1. * p < 0.01. ** p < 0.05. Dunnett’s test. Samples for gene expression analysis were analyzed in triplicates and the scores are presented as means ± SD

Article Snippet: Membranes were either incubated with anti-human Notch2 intracellular domain antibody (R&D systems, MN, USA; 1:2000), anti-FLAG M2 monoclonal antibody (Sigma-Aldrich; 1:1000) or anti-Actin antibody clone C4 (Merck Millipore; 1:10,000) overnight at 4 °C.

Techniques: Western Blot, Positive Control, Expressing, Cell Culture, Gene Expression, Transfection, Plasmid Preparation, Dominant Negative Mutation

Journal: BMC Veterinary Research

Article Title: Notch2 signal is required for the maintenance of canine hemangiosarcoma cancer stem cell-like cells

doi: 10.1186/s12917-018-1624-8

Figure Lengend Snippet: a Colony numbers of HSA cell lines overexpressing Notch2 vector constructs. b (Top) Representative images of JuB2 and Ud6, 5 and 7 days after starting serum-free culture, respectively. Bars = 100 μm. (Bottom) Relative cell numbers of JuB2 and Ud6. Number of Vec was set to 1. * p < 0.01. ** p < 0.05. Dunnett’s test. The scores are presented as means ± SD

Article Snippet: Membranes were either incubated with anti-human Notch2 intracellular domain antibody (R&D systems, MN, USA; 1:2000), anti-FLAG M2 monoclonal antibody (Sigma-Aldrich; 1:1000) or anti-Actin antibody clone C4 (Merck Millipore; 1:10,000) overnight at 4 °C.

Techniques: Plasmid Preparation, Construct

Journal: BMC Veterinary Research

Article Title: Notch2 signal is required for the maintenance of canine hemangiosarcoma cancer stem cell-like cells

doi: 10.1186/s12917-018-1624-8

Figure Lengend Snippet: Immunohistochemistry analysis for clinical HSA cases using anti-human Notch2 antibody. Insertion indicated the magnified views of tumor cells or normal endothelial cells. Arrows = neoplastic cells. Arrow heads = endothelial cells in normal blood vessels in the same slides. Asterisks = lymphocytes. Bars = 50 μm

Article Snippet: Membranes were either incubated with anti-human Notch2 intracellular domain antibody (R&D systems, MN, USA; 1:2000), anti-FLAG M2 monoclonal antibody (Sigma-Aldrich; 1:1000) or anti-Actin antibody clone C4 (Merck Millipore; 1:10,000) overnight at 4 °C.

Techniques: Immunohistochemistry

Journal: BMC Veterinary Research

Article Title: Notch2 signal is required for the maintenance of canine hemangiosarcoma cancer stem cell-like cells

doi: 10.1186/s12917-018-1624-8

Figure Lengend Snippet: Primer list for RT-qPCR

Article Snippet: Membranes were either incubated with anti-human Notch2 intracellular domain antibody (R&D systems, MN, USA; 1:2000), anti-FLAG M2 monoclonal antibody (Sigma-Aldrich; 1:1000) or anti-Actin antibody clone C4 (Merck Millipore; 1:10,000) overnight at 4 °C.

Techniques: Sequencing