Journal: British Journal of Cancer

Article Title: Antiapoptotic effect of haem oxygenase-1 induced by nitric oxide in experimental solid tumour

doi: 10.1038/sj.bjc.6600830

Figure Lengend Snippet: Apoptosis induction and change in HO activity after treatment with ZnPP IX of AH136B solid. Apoptosis ( A, B ) and HO activity ( C ) were assessed on day 14 after tumour implantation. Each specimen was analysed via TUNEL staining 24 h after treatment with vehicle (the control) ( A, a ), with ZnPP IX (500 μ g kg −1 i.a.) ( A, b ), or with CuPP IX (500 μ g kg −1 i.a.) ( A, c ). Quantitative analysis of TUNEL-positive cells in each specimen is shown ( B ). TUNEL-positive cells were counted in four different fields of magnification at × 100 per sample, and then the number of positive cells per mm 2 was calculated. Haem oxygenase activity of the solid tumour was measured after treatment in the same manner as in the TUNEL analysis ( C ). ** P <0.05, ** P <0.01 vs control ( n =3 for each group). Data are means±s.e. See text for details.

Article Snippet: AH136B tumour (rat hepatoma) cells were implanted subcutaneously (s.c.) in a dorsal site on the foot of male Donryu rats weighing 160–180 g (SLC, Inc., Shizuoka, Japan) with an inoculum size of 1 × 10 7 cells per injection site as described previously ( Doi et al , 1996 ).

Techniques: Activity Assay, TUNEL Assay, Staining, Control

Journal: British Journal of Cancer

Article Title: Antiapoptotic effect of haem oxygenase-1 induced by nitric oxide in experimental solid tumour

doi: 10.1038/sj.bjc.6600830

Figure Lengend Snippet: Zinc protoporphyrin IX-induced apoptosis of AH136B cells in vitro . AH136B cells were incubated for 24 h with indicated concentrations of ZnPP IX, with or without caspase-3 inhibitor ( A ) or SnPP IX ( B ), or with 100 μ M CuPP IX. ( A, B ) TUNEL-positive cells were counted in four different fields of magnification at × 100 per sample, and then the percentage of positive cells was calculated. ** P <0.05 vs control ( n =3 for each group). Cell viability after the same treatment is shown in the inset. ** P < 0.05, ** P <0.01 vs control ( n =4 for each group). Data are means±s.e. ( C ) Representative TUNEL staining of control and ZnPP IX-treated tumour cells. See text for details.

Article Snippet: AH136B tumour (rat hepatoma) cells were implanted subcutaneously (s.c.) in a dorsal site on the foot of male Donryu rats weighing 160–180 g (SLC, Inc., Shizuoka, Japan) with an inoculum size of 1 × 10 7 cells per injection site as described previously ( Doi et al , 1996 ).

Techniques: In Vitro, Incubation, TUNEL Assay, Control, Staining

Journal: British Journal of Cancer

Article Title: Antiapoptotic effect of haem oxygenase-1 induced by nitric oxide in experimental solid tumour

doi: 10.1038/sj.bjc.6600830

Figure Lengend Snippet: Effect of ZnPP IX on HO activity and caspase-3 activity of AH136B cells cultured in vitro . Haem oxygenase activity was measured 24 h after incubation with vehicle (control), ZnPP IX, or CuPP IX ( A ). Similarly, cells were treated with 100 μ M ZnPP with or without caspase-3 inhibitor (1 or 10 μ M ) for 24 h ( B ). Caspase-3 activity was measured fluorome-trically in cell extracts by using a fluorescent substrate. ** P <0.05, ** P <0.01 vs control ( n =3 for each group). Data are means±s.e. See text for details.

Article Snippet: AH136B tumour (rat hepatoma) cells were implanted subcutaneously (s.c.) in a dorsal site on the foot of male Donryu rats weighing 160–180 g (SLC, Inc., Shizuoka, Japan) with an inoculum size of 1 × 10 7 cells per injection site as described previously ( Doi et al , 1996 ).

Techniques: Activity Assay, Cell Culture, In Vitro, Incubation, Control

Journal: British Journal of Cancer

Article Title: Antiapoptotic effect of haem oxygenase-1 induced by nitric oxide in experimental solid tumour

doi: 10.1038/sj.bjc.6600830

Figure Lengend Snippet: Protective effect of bilirubin against ZnPP IX-induced apoptosis of AH136B cells. AH136B cells were incubated for 24 h with 100 μ M ZnPP IX in the presence or absence of indicated concentrations of bilirubin. TUNEL-positive cells were counted in four different fields of magnification at × 100 per sample, and then the percentage of positive cells was calculated. ** P <0.05 vs ZnPP IX alone ( n =3 for each group). Data are means±s.e. See text for details.

Article Snippet: AH136B tumour (rat hepatoma) cells were implanted subcutaneously (s.c.) in a dorsal site on the foot of male Donryu rats weighing 160–180 g (SLC, Inc., Shizuoka, Japan) with an inoculum size of 1 × 10 7 cells per injection site as described previously ( Doi et al , 1996 ).

Techniques: Incubation, TUNEL Assay

Journal: British Journal of Cancer

Article Title: Antiapoptotic effect of haem oxygenase-1 induced by nitric oxide in experimental solid tumour

doi: 10.1038/sj.bjc.6600830

Figure Lengend Snippet: Tumour blood flow after ZnPP IX or L -NAME treatment. Tumour blood flow was measured in AH136B solid tumour on day 14 after tumour implantation. At the indicated times after injection of 500 μ g kg −1 ZnPP IX (i.a.) or 6 mg kg −1 L -NAME (i.p.), a laser Doppler flowmeter and a probe needle were used to measure blood flow. Relative changes in tumour blood flow after injection of ZnPP IX or L -NAME are shown. Inset, change in tumour blood flow during early period after treatment (within 25 min). ** P <0.01 by ANOVA between the groups ( n =3 for each time point). Data are means±s.e. See text for details.

Article Snippet: AH136B tumour (rat hepatoma) cells were implanted subcutaneously (s.c.) in a dorsal site on the foot of male Donryu rats weighing 160–180 g (SLC, Inc., Shizuoka, Japan) with an inoculum size of 1 × 10 7 cells per injection site as described previously ( Doi et al , 1996 ).

Techniques: Injection

Journal: British Journal of Cancer

Article Title: Antiapoptotic effect of haem oxygenase-1 induced by nitric oxide in experimental solid tumour

doi: 10.1038/sj.bjc.6600830

Figure Lengend Snippet: Western blot analysis of HSP70 and HO-1 proteins in AH136B cells and solid tumours. ( A ) Cells were incubated with SNAP (10 or 100 μ M ), P-NONOate (10 or 100 μ M ), or ZnPP IX (100 μ M ) for 6 h or, as a positive control, were treated by heat at 42°C for 30 min, followed by incubation at 37°C for 6 h. ( B ) AH136B solid tumours were treated with 0.9% NaCl solution (control), ZnPP IX (500 μ g kg −1 i.a.), L -NAME (6 mg kg −1 day −1 for 5 days i.p.), or SMT (6 mg kg −1 day −1 for 5 days i.p.). ( C ) Solid tumours were resected at 24 or 48 h after initiation of surgical occlusion of the common iliac artery, which serves the tumour-feeding artery of the tumour-implanted side. Control rats were treated by sham operation. Then, expression of HSP70 and HO-1 proteins was detected by Western blotting by using a monoclonal antibody to mammalian inducible HSP70 or a polyclonal antibody to rat HO-1. Each data point shown represents three independent experiments. See text for details.

Article Snippet: AH136B tumour (rat hepatoma) cells were implanted subcutaneously (s.c.) in a dorsal site on the foot of male Donryu rats weighing 160–180 g (SLC, Inc., Shizuoka, Japan) with an inoculum size of 1 × 10 7 cells per injection site as described previously ( Doi et al , 1996 ).

Techniques: Western Blot, Incubation, Positive Control, Control, Expressing

Journal: Nature Communications

Article Title: Pancreatic tumor eradication via selective Pin1 inhibition in cancer-associated fibroblasts and T lymphocytes engagement

doi: 10.1038/s41467-022-31928-7

Figure Lengend Snippet: a Establishment and validation of CAFs via high expressions of both FAP-α and α-SMA. b Cellular uptake of Cy5-labeled formulations on Pan02 cells, NIH-3T3 cells, and CAFs, measured by flow cytometry ( n = 3 biologically independent experiments; error bars, mean ± SD). c Cellular uptake of Cy5-labeled antiCAFs-DMS on cells, imaged by fluorescent microscopy. Scale bars are 20 μm. Three independent repetitions got similar results. d Cellular uptake of Cy5-labeled antiCAFs-DMS on cells, quantified by flow cytometry ( n = 6 biologically independent samples; error bars, mean ± SD). Cytotoxicity on Pan02 cells ( e ) and CAFs ( f ) ( n = 3 biologically independent experiments; error bars, mean ± SD). g Western blotting detection of Pin1 and relative proteins from Pan02 cells after treating with PBS, AG17724 (0.5 μM), or antiCAFs-DMS (corresponds to 0.5 μM of AG17724) for 24 hours. h Quantitative analysis of the western blotting results from Pan02 cells (Three independent repetitions got similar results.). i Western blotting detection of Pin1 and relative proteins from CAFs treated with PBS, AG17724, or antiCAFs-DMS after treating with PBS, AG17724 (0.5 μM) or antiCAFs-DMS (corresponds to 0.5 μM of AG17724) for 24 hours. j Quantitative analysis of the western blotting results from CAFs (Three independent repetitions got similar results.). One-way analysis of variance followed by Turkey posttests. Source data are provided as a Source Data file.

Article Snippet: Anti-FAP-α antibody (MAB9727-100, 0.25 μg/mL as working concentration.) and anti-phospho-CDC2/CDK1 (Y15) antibody (AF888-SP, 0.2 μg/mL as working concentration.) were purchased from R&D SYSTEMS.

Techniques: Biomarker Discovery, Labeling, Flow Cytometry, Microscopy, Western Blot

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: IGFBP-rP1-knockdown in RF/6A cells by siIGFBP-rP1. (A) Reverse transcription-quantitative PCR analysis of IGFBP-rP1 transcript expression in RF/6A cells. GAPDH served as an internal reference for control. Both siIGFBP-rP1 duplex 1 and 2 significantly inhibited the expression IGFBP-rP1 compared with controls ( # P<0.05). Furthermore, the inhibitory effect of siIGFBP-rP1 duplex 2 was significantly increased compared with siIGFBP-rP1 duplex 1 (*P<0.05). (B) IGFBP-rP1 expression was measured by western blotting and normalized to that of β-actin. The inhibitory effect of siIGFBP-rP1 on the IGFBP-rP1 protein expression was consistent with that of the RNA expression. The membranes were stripped off and probed for the proteins. Data are presented as the mean ± standard deviation of three independent experiments with similar results and calculated as the integrated optical density of IGFBP-rP1 relative to the internal reference. # P<0.01 vs. the blank control group. *P<0.05 vs. the siIGFBP-rP1 duplex 1 group. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific siRNA. Lane M, marker; lane 1, blank control; lane 2, transfection reagent; lane 3, scrambled control siRNA; lane 4, siRNA duplex 1; lane 5, siRNA duplex 2.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Knockdown, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Control, Western Blot, RNA Expression, Standard Deviation, Binding Assay, Marker, Transfection

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: Cell growth curves of siIGFBP-rP1-transfected cells and untransfected cells cultured in normoxic conditions for 6, 12, 24, 48 and 72 h. The OD values of transfected cells at 12, 24 and 48 h were significantly higher compared with untransfected cells (*P<0.01). Furthermore, the OD value of siIGFBP-rP1 duplex 2-transfected cells was significantly higher compared with siIGFBP-rP1 duplex 1-transfected cells at 24 h ( # P<0.01). No significant differences were observed among the groups at 6 and 72 h. OD values are presented as the mean ± standard deviation of 4 wells/group and experiments were performed in triplicate. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific siRNA; OD, optical density.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Transfection, Cell Culture, Standard Deviation, Binding Assay

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: Cell growth curves of siIGFBP-rP1-transfected or untransfected cells cultured under normoxic or hypoxic conditions for 6, 12, 24, 48 and 72 h, as detected by MTS colorimetric assays. The OD values of hypoxic groups (CoCl 2 and 1% O 2 ) decreased significantly at 12, 24, 48 and 72 h compared with the control group ( # P<0.01). There was no significant difference between the hypoxic groups (P>0.05). The OD value of the siRNA group were significantly increased at 12, 24 and 48 h compared with controls (**P<0.01). Furthermore, the OD values of the transfected cells cultured in hypoxic conditions (CoCl 2 + siRNA group and 1% O 2 + siRNA group) for 12, 24, 48 and 72 h were significantly lower compared with the siRNA group; additionally, the values were significantly higher compared with the hypoxic groups (*P<0.01). No significant differences were identified between the CoCl 2 + siRNA, 1% O 2 + siRNA and control groups (P>0.05). OD values are presented as the mean ± standard deviation of 4 wells/group and experiments were performed in triplicate. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific siRNA; CoCl 2 , cobalt chloride; OD, optical density.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Transfection, Cell Culture, Control, Standard Deviation, Binding Assay

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: Cell motility of siIGFBP-rP1-transfected or untransfected cells cultured under normoxic or hypoxic conditions for 24 h is detected by wound and Transwell assays. (A) Representative images showing that RF/6A cells migrated across the wound boundary to the blank area (light microscopy; magnification, ×400) and (B) passed across the filter toward the lower surface (light microscopy; magnification, ×200). Hypoxia significantly promoted cell mobility compared with the controls ( # P<0.01). siIGFBP-rP1 transfection (siIGFBP-rP1 group) further enhanced cell migration, as compared with untransfected cells cultured in hypoxic conditions (hypoxia group; *P<0.01). The transfected cells cultured in hypoxic conditions (hypoxia + siRNA group) exhibited a significantly increased migration ability compared with the transfected cells cultured in normoxic conditions (siIGFBP-rP1 group; **P<0.01). Values are presented as the mean ± standard deviation of 4 samples/group and experiments were performed in triplicate and are quantified as the ratio relative to the control group. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific siRNA.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Transfection, Cell Culture, Light Microscopy, Migration, Standard Deviation, Control, Binding Assay

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: IGFBP-rP1-silencing stimulates hypoxia-induced tube formation of RF/6A cells. Representative images (inverted phase contrast microscopy; magnification, ×50) demonstrating that RF/6A cells formed capillary-like tube structures within the Matrigel layer in different mediums. RF/6A cells in hypoxic conditions or siIGFBP-rP1-transfected cells significantly formed completely enclosed capillary-like tubes compared with the controls ( # P<0.01 the hypoxia group vs. the control group; *P<0.01 the siIGFBP-rP1 group vs. the control group). Moreover, siIGFBP-rP1 transfection further promoted tube formation in RF/6A cells in the hypoxia + siIGFBP-rP1 group compared with the siIGFBP-rP1 group (**P<0.01). The values are presented as the mean ± standard deviation of 4 samples/group and experiments were performed in triplicate and are quantified as the ratio relative to the control group. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific small interfering RNA.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Microscopy, Transfection, Control, Standard Deviation, Binding Assay, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: IGFBP-rP1-silencing upregulates the hypoxia-induced RAF/MEK/ERK signaling pathway activation and VEGF expression. Representative images and quantified data demonstrated that hypoxic stress upregulated B-RAF, p-MEK, p-ERK and VEGF expression in RF/6A cells compared with controls ( # P<0.05). siIGFBP-rP1 transfection significantly promoted hypoxia-induced B-RAF, p-MEK, p-ERK and VEGF expression in RF/6A compared with the hypoxia group ( # P<0.05). IGFBP-rP1 restoration significantly downregulated the expression of B-RAF, p-MEK, p-ERK and VEGF in siIGFBP-rP1-transfected cells, under both normoxic and hypoxic conditions, compared with the siIGFBP-rP1 and hypoxia + siIGFBP-rP1 groups, respectively (*P<0.01). The membranes were stripped off and probed for the proteins. Values are presented as the mean ± SD of 3 independent experiments with similar results and are presented as the integrated optical density of studied proteins relative to GAPDH. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific siRNA. Lane 1, control; lane 2, siIGFBP-rP1; lane 3, siIGFBP-rP1 + IGFBP-rP1; lane 4, hypoxia; lane 5, hypoxia + siIGFBP-rP1; lane 6, hypoxia + siIGFBP-rP1 + IGFBP-rP1.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Activation Assay, Expressing, Transfection, Binding Assay, Control

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