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: Pathology

Article Title: Pathobiology of Notch2 in lung cancer.

doi: 10.1016/j.pathol.2017.05.005

Figure Lengend Snippet: Fig. 1 Immunohistochemical analysis of NOTCH receptors and CC10 expression in non-neoplastic lungs. Serial lung section stained for (A) Notch1, (B) Notch2, and (C) CC10. (A) The surface of bronchiolar (arrow) and alveolar epithelial cells (arrowhead) was positively stained for Notch1. (B) Bronchiolar epithelial cells (arrow) were positive for Notch2, but alveolar epithelial cells (arrowhead) were not. (C) The cytoplasm of Club cells was positive for CC10. Scale bar = 20 mm.

Article Snippet: We purchased Notch2 siRNA (sc-40135) and control siRNA (sc-36869) from Santa Cruz Biotechnology (Santa Cruz, USA) and transfected them into cells using an electroporator as previously described.20

Techniques: Immunohistochemical staining, Expressing, Staining

Journal: Pathology

Article Title: Pathobiology of Notch2 in lung cancer.

doi: 10.1016/j.pathol.2017.05.005

Figure Lengend Snippet: Fig. 2 Evaluation of NOTCH receptors and CC10 expression in lung cancer. (A) Western blot analysis to detect Notch1, Notch2, and Notch3 receptors was performed using 13 human lung cancer cell lines, including seven SCLC, three ADC, and three SCC cell lines. b-actin served as an internal control. (B) Upper panel: Most adenocarcinoma tissues were positive for Notch2, but negative for CC10. Lower panel: A few adenocarcinoma tissues were positive for both of Notch2 and CC10. Scale bar = 20 mm. (C) Quantitative real-time polymerase chain reaction analysis showed that transient transfection of the Notch2 intracellular domain (N2ICD) gene in H358 cells results in up-regulation of Notch2 (left) and CC10 (right) mRNA. Each bar shows relative expression level compared to expression level in the cells transfected mock plasmid. * p < 0.001, ** p < 0.05. (D) ICC study of H358 cells transfected with mock and N2ICD genes. GFP positive cells transfected with mock were negative for CC10, but N2ICD transfection induced CC10. Scale bar = 20 mm.

Article Snippet: We purchased Notch2 siRNA (sc-40135) and control siRNA (sc-36869) from Santa Cruz Biotechnology (Santa Cruz, USA) and transfected them into cells using an electroporator as previously described.20

Techniques: Expressing, Western Blot, Control, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation

Journal: Pathology

Article Title: Pathobiology of Notch2 in lung cancer.

doi: 10.1016/j.pathol.2017.05.005

Figure Lengend Snippet: Fig. 3 Western blotting analysis of Notch2 knockdown in human lung cancer cell lines. Western blotting analysis was performed using lung cancer cell lines, including adenocarcinoma (A549, H358), squamous cell carcinoma (H226), and small cell lung carcinoma (SBC3, H69AR). Three biological replicates were analysed. Repre- sentatives of no treat (NT), transfected control siRNA (cont.), and Notch2 siRNA (siN2) are shown. Expressions of phospho-histone H3 (PHH3) as a mitotic marker, caspase3 (CASP3), cleaved caspase3 (cCASP3), B-cell lymphoma 2 (Bcl-2), and phospho-Bcl-2 (pBcl-2) as apoptosis markers, AKT and phospho-AKT (pAKT) as cell signal pathway activity markers, chromogranin A (CGA), synaptophysin (SYP), neural cell adhesion molecule (NCAM), calcitonin gene-related peptide (CGRP), and alpha-subunit of guanine nucleotide-binding protein Go (Goa) as neuroendocrine markers in lung cancer cell lines are presented. Notch2 knockdown was successfully performed, and all markers showed similar expression patterns between control siRNA and Notch2 siRNA in non-small cell cancer cell lines. Notch2 siRNA induced suppression of pAKT in SBC3, and increase of cCASP3 in H69AR. b-actin served as an internal control.

Article Snippet: We purchased Notch2 siRNA (sc-40135) and control siRNA (sc-36869) from Santa Cruz Biotechnology (Santa Cruz, USA) and transfected them into cells using an electroporator as previously described.20

Techniques: Western Blot, Knockdown, Transfection, Control, Marker, Activity Assay, Binding Assay, Expressing

Journal: Pathology

Article Title: Pathobiology of Notch2 in lung cancer.

doi: 10.1016/j.pathol.2017.05.005

Figure Lengend Snippet: Fig. 4 Mutual relationship among NOTCH receptors. (A) Western blot analysis was performed to evaluate the mutual effect of siNotch1, siNotch2, and siNotch3 in A549 and H69AR cell lines. Three biological replicates were analysed. Notch1 knockdown decreased Notch2 and Notch3. Notch2 knockdown did not change the expression of Notch1 and Notch3, but increased the Notch1 intracellular domain (N1ICD). Notch3 knockdown increased Notch1 but not Notch2. b-actin served as an internal control. (B) Quantitative real-time polymerase chain reaction analysis of Notch1 and Notch3 in H358 cells transiently transfected with the Notch2 intracellular domain gene (trN2). Transfection of the Notch2 intracellular domain significantly decreased Notch1 expression, but not Notch3. Each bar shows relative expression level compared to expression level in the cells transfected mock plasmid. * p < 0.05. (C) Notch1 receives positive feedback from Notch3, and negative feedback from Notch2. Notch2 and Notch3 did not affect each other.

Article Snippet: We purchased Notch2 siRNA (sc-40135) and control siRNA (sc-36869) from Santa Cruz Biotechnology (Santa Cruz, USA) and transfected them into cells using an electroporator as previously described.20

Techniques: Western Blot, Knockdown, Expressing, Control, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation

Journal: Frontiers in Oncology

Article Title: Cooperation Between Cancer and Fibroblasts in Vascular Mimicry and N2-Type Neutrophil Recruitment via Notch2–Jagged1 Interaction in Lung Cancer

doi: 10.3389/fonc.2021.696931

Figure Lengend Snippet: Interaction of Notch2 and Jagged1 between CAFs and lung cancer cells. (A) Expression of Notches and their ligands in lung cancer cells and their CAFs, respectively. (B) Close interaction of Nocth2 and Jagged1 between H1299 cells and CAFs as determined by proximity ligation assay (PLA). H1299-GFP cells, CAFs (BioTracker 400 Blue cytoplasmic membrane dye), and H1299-GFP cells mixed with CAFs were committed to PLA analysis (red). (C) Increased NOTCH 2 intracellular domain (N2ICD) nuclear translocation in H1299 cells. Cells were collected and the nuclear and cytosol fractions were extracted using a commercial kit. Equal levels of protein were assessed by immunoblotting. (D) Inhibition of Jagged1 in CAFs reduced the VM formation of H1299 cells. Knockdown of Jagged 1 in CAFs reduced (E) N2ICD nuclear translocation, and (F) HEY1 upregulation driven by CAFs in H1299 cells. Knockdown of Notch2 in H1299 reduced (G) VM formation, (H) N2ICD nuclear translocation, and (I) HEY1 upregulation driven by CAFs. CAFs and H1299 cells were transfected with a control, Jagged1 (for CAFs), or Notch2 (for H1299 cells) siRNA for 24 h, then CAFs were mixed with H1299 cells. The HEY1 mRNA and nuclear N2ICD levels were detected using a quantitative reverse transcription polymerase chain reaction and immunoblotting, respectively. All results are representative of at least three independent experiments. Each value is the mean ± SD of three determinations; *P < 0.05.

Article Snippet: N2ICD (catalog no. AF3735) was obtained from R&D system.

Techniques: Expressing, Proximity Ligation Assay, Membrane, Translocation Assay, Western Blot, Inhibition, Knockdown, Transfection, Control, Reverse Transcription, Polymerase Chain Reaction