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: Journal of Experimental & Clinical Cancer Research : CR

Article Title: The Hippo pathway effector TAZ induces intrahepatic cholangiocarcinoma in mice and is ubiquitously activated in the human disease

doi: 10.1186/s13046-022-02394-2

Figure Lengend Snippet: Primary antibodies used for immunohistochemistry (IHC) and Western blot analysis (WB)

Article Snippet: Notch2 , IHC , 1:50 , Cell Signaling Technology , 5732.

Techniques: Immunohistochemistry, Western Blot, Concentration Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: The Hippo pathway effector TAZ induces intrahepatic cholangiocarcinoma in mice and is ubiquitously activated in the human disease

doi: 10.1186/s13046-022-02394-2

Figure Lengend Snippet: Validated Gene Expression Assays used for real-time qRT-PCR experiments

Article Snippet: Notch2 , IHC , 1:50 , Cell Signaling Technology , 5732.

Techniques: Gene Expression

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: The Hippo pathway effector TAZ induces intrahepatic cholangiocarcinoma in mice and is ubiquitously activated in the human disease

doi: 10.1186/s13046-022-02394-2

Figure Lengend Snippet: Liver lesions developed in AKT/TAZ mice display activation of Hippo and AKT/mTOR pathways. ( A ) Representative immunohistochemical patterns of a cholangiocellular tumor exhibiting immunoreactivity for HA-Tag(AKT) and nuclear TAZ, as well as for downstream effectors of the Hippo (NOTCH1, NOTCH2, and JAG1) and AKT/mTOR (phosphorylated/activated AKT or p-AKT; phosphorylated/inactivated GSK-3β or p-GSK-3β; and phosphorylated/activated RPS6 or p-RPS6) pathways. Original magnification: 200x; scale bar: 100 μm. ( B ) Upregulation of the Hippo pathway targets CCN1 , CCN2 , and NOTCH 2 in AKT/TAZ livers (10 weeks post-injection) compared with livers injected with the empty vector (Vector), as assessed by quantitative real-time RT-PCR. Abbreviations: H&E, hematoxylin and eosin staining; ST, non-tumorous surrounding tissue; T, tumor. Data are expressed as means ± SD. *** p < 0.0001 and ** p < 0.01 vs. empty vector-injected mice

Article Snippet: Notch2 , IHC , 1:50 , Cell Signaling Technology , 5732.

Techniques: Activation Assay, Immunohistochemical staining, Injection, Plasmid Preparation, Quantitative RT-PCR, Staining

Journal: International Journal of Molecular Sciences

Article Title: Zinc Finger 521 Modulates the Nrf2-Notch Signaling Pathway in Human Ovarian Carcinoma

doi: 10.3390/ijms241914755

Figure Lengend Snippet: Correlation analysis between ZNF521 and the NRF2-NOTCH axis in hOC. R2 analysis of 381 specimens in a hOC public database (Tumor Ovarian Serous Cystoadenocarcinoma 2022-v32): ZNF521 expression data plotted vs. those of P300, NRF2 (NFE2L2 in figure), NOTCH1 and NOTCH2 ( A ); P300 expression data plotted vs. those of NRF2 (NFE2L2 in figure), NOTCH1 and NOTCH2 ( B ).

Article Snippet: ZNF521 was detected using a rabbit anti-ZNF521 (PA534388, Life Technologies) antibody at 1:1000, P300 with a rabbit anti-P300(C-20) (sc-585, Santa Cruz Biotechnology) antibody at 1:10000, NRF2 with a rabbit anti-NRF2 (D1Z9C) XP (#12721, Cell Signaling) antibody, NOTCH1 with a rabbit anti-NOTCH1 (ab-52301, AbCam, Milan, Italy) antibody at 1:500, NOTCH2 with a rabbit anti-NOTCH2 (sc-5545, Santa Cruz Bio-technology) antibody at 1:1000, P53 with a rabbit anti-P53 (#9282, Cell Signaling) antibody at 1:1000 and GADPH with a mouse anti-GAPDH (sc-166574 Santa Cruz Biotechnology) antibody at 1:1000.

Techniques: Expressing

Journal: Journal of Personalized Medicine

Article Title: Dysregulated Notch Signaling in the Airway Epithelium of Children with Wheeze

doi: 10.3390/jpm11121323

Figure Lengend Snippet: Notch receptors gene expression profiles in ex vivo pAEC from children with and without wheeze. Relative expression of NOTCH1–4 receptors ( A – D ) were measured in ex vivo pAEC from children with and without wheeze ( n ≥ 6) by real-time qPCR (refer to 2.5.4). ( A ) NOTCH1 was the most abundantly expressed Notch receptor gene in ex vivo pAEC from a paediatric cohort. However, NOTCH1 mRNA expression was not significantly different between pAEC from children without wheeze. ( B ) In contrast, mRNA expression of NOTCH2 was significantly lower in pAEC from children with wheeze compared to their non-wheeze counterparts. ( C , D ) Finally, no significant differences were observed between pAEC from children with and without wheeze in mRNA expression of NOTCH3 and NOTCH4, although the genes were expressed at different magnitudes. Each dot represents the mean of two replicates from a single ex vivo sample. Data are normalized to housekeeping gene, 18S and represented as median ± IQR. * Statistical significance relative to the non-wheeze cohort ( p < 0.050; Mann–Whitney U-test).

Article Snippet: Antibodies against human Notch2 extracellular (monoclonal rabbit, clone D67C8, #4530; Cell Signaling Technologies, Danvers, MA, USA) and intracellular (monoclonal mouse, clone 602845, MAB37351; R&D Systems) domains, Jagged1 (polyclonal goat, AF1277; R&D Systems), were also obtained.

Techniques: Gene Expression, Ex Vivo, Expressing, MANN-WHITNEY

Journal: Journal of Personalized Medicine

Article Title: Dysregulated Notch Signaling in the Airway Epithelium of Children with Wheeze

doi: 10.3390/jpm11121323

Figure Lengend Snippet: NOTCH2 and JAG1 gene expression profiles in pAEC post in vitro wounding. Relative expression of NOTCH2 ( A ) and JAG1 ( B ) mRNA were measured in pAEC post in vitro wounding from children with ( n = 6) and without wheeze ( n = 6) by real-time qPCR. ( A , B ) Following wounding, pAEC from children without wheeze displayed a significant upregulation of NOTCH2 and JAG1 mRNA expression at 48 h post wounding respectively. By 72 h post wounding, NOTCH2 mRNA expression ( A ) had returned to baseline levels, whilst JAG1 mRNA expression ( B ) was maintained at about 3-fold higher levels than baseline. In contrast, pAEC cultures from children with wheeze displayed lower gene expression levels of NOTCH2 (A) and higher gene expression levels of JAG1 ( B ) at baseline. An earlier induction of NOTCH2 mRNA expression was observed in pAEC from children with wheeze at 24 h post wounding, which was sustained to 72 h post wounding. Although JAG1 mRNA levels were induced by 24 h post wounding, JAG1 expression returned to baseline levels by 72 h post wounding indicating distinct differences in the kinetics of Notch signaling during airway epithelial repair in children with wheeze. Data are represented as median ± IQR. * Statistical significance of the wheeze group relative to matching non-wheeze timepoint sample ( p < 0.050; Mann–Whitney U-test), or # statistical significance of relevant timepoint to time 0 h of matching patient cohort ( p < 0.050; Kruskal–Wallis test with Dunn’s test for multiple comparisons).

Article Snippet: Antibodies against human Notch2 extracellular (monoclonal rabbit, clone D67C8, #4530; Cell Signaling Technologies, Danvers, MA, USA) and intracellular (monoclonal mouse, clone 602845, MAB37351; R&D Systems) domains, Jagged1 (polyclonal goat, AF1277; R&D Systems), were also obtained.

Techniques: Gene Expression, In Vitro, Expressing, MANN-WHITNEY

Journal: Journal of Personalized Medicine

Article Title: Dysregulated Notch Signaling in the Airway Epithelium of Children with Wheeze

doi: 10.3390/jpm11121323

Figure Lengend Snippet: Blocking of Notch signaling in pAEC of children without wheeze. ( A ) Monolayer pAEC cultures from children without wheeze were treated with increasing concentrations (1 nM–10 μM; green circles, triangles, squares) of the GSI, DAPT 24 h prior to wounding. Wound closure rates of pAEC from children without wheeze treated with DAPT, DMSO vehicle (black diamonds) (0.1% v / v ) or untreated negative control (blue circles) were determined. Full wound closure in DMSO vehicle and untreated negative controls were achieved by approximately 54 h post-wounding. Cultures treated with DAPT showed a concentration-dependent reduction in wound closure and full wound closure was not achieved within the completion of the experiment at 72 h post-wounding. Representative phase-contrast images of DMSO vehicle control (0.1% v / v ) and DAPT (10 µM) treated cultures from children without wheeze at 72 h post-wounding. ( B ) Monolayer pAEC cultures from children without wheeze were treated with either 0.1 (green squares) or 10 (green triangles) µg/mL Notch2 blocking antibody 24 h prior to and during wounding. Notch2 blocking (10 µg/mL; green triangles) resulted in reduced wound closure compared to the negative and isotype antibody controls (10 µg/mL). Representative phase-contrast images of cultures from children without wheeze treated with either isotype antibody control or Notch2 antibody (10 µg/mL) at 72 h post wounding. ( C ) Monolayer pAEC cultures from children with wheeze were treated with either 0.1 (purple squares) or 10 (purple triangles) µg/mL Jagged1 blocking antibody 24 h prior to and during wounding. Jagged1 blocking (10 µg/mL) resulted in a marginal reduction in wound closure compared to the negative and isotype antibody controls (10 µg/mL). Representative phase-contrast images of cultures from children without wheeze treated with either isotype antibody control or Jagged1 antibody (10 µg/mL) at 72 h post wounding. Experiments were performed in cultures from children with ( n = 6) or without wheeze ( n = 5). Data are represented as mean ± SD. * Statistical significance relative to DMSO vehicle or isotype antibody controls ( p < 0.050, Friedman test with Dunn’s multiple comparisons test). Dashed lines in phase-contrast images indicate the original wound area at t = 0 h post-wounding. Scale bar is 200 µm (10× objective).

Article Snippet: Antibodies against human Notch2 extracellular (monoclonal rabbit, clone D67C8, #4530; Cell Signaling Technologies, Danvers, MA, USA) and intracellular (monoclonal mouse, clone 602845, MAB37351; R&D Systems) domains, Jagged1 (polyclonal goat, AF1277; R&D Systems), were also obtained.

Techniques: Blocking Assay, Negative Control, Concentration Assay, Control

Journal: Journal of Personalized Medicine

Article Title: Dysregulated Notch Signaling in the Airway Epithelium of Children with Wheeze

doi: 10.3390/jpm11121323

Figure Lengend Snippet: Overexpression of Notch2 in pAEC from children with wheeze. ( A – D ) Cultures from children with wheeze were infected with a retrovirus containing either the empty vector-GFP or Notch2-GFP. ( A ) Protein expression of Notch2 was measured by In-Cell Western and normalized to cell number. ( B ) Immunofluorescent staining for Notch2 intracellular domain protein identified its nuclear staining in Notch2-overexpressed cultures. Scale bar: 200 µm (20× magnification) ( C ) Monolayer pAEC cultures expressing Notch2-GFP (purple triangles) or empty vector-GFP (orange squares) and wildtype controls (red circles) displayed comparable repair capacity following in vitro scratch wounding. ( D ) Cultures from children with wheeze (red circles) showed significantly lower cell proliferation rates compared to non-wheeze controls (blue circles). Notch2 overexpression (purple triangles) had no effect on cell proliferation compared to empty vector-GFP (orange squares) or wildtype wheeze controls (red circles). Experiments utilized pAEC cultures from children with ( n = 6) and without wheeze ( n = 5). Data are represented as median ± IQR (* p < 0.050, wheeze vs. non-wheeze; # p < 0.050, empty vector/Notch2 vs. Wildtype control; Dunn’s multiple comparisons test).

Article Snippet: Antibodies against human Notch2 extracellular (monoclonal rabbit, clone D67C8, #4530; Cell Signaling Technologies, Danvers, MA, USA) and intracellular (monoclonal mouse, clone 602845, MAB37351; R&D Systems) domains, Jagged1 (polyclonal goat, AF1277; R&D Systems), were also obtained.

Techniques: Over Expression, Infection, Plasmid Preparation, Expressing, In-Cell ELISA, Staining, In Vitro, Control

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

Journal: British Journal of Cancer

Article Title: Notch2 pathway mediates breast cancer cellular dormancy and mobilisation in bone and contributes to haematopoietic stem cell mimicry

doi: 10.1038/s41416-019-0501-y

Figure Lengend Snippet: Spindle-shaped N-cadherin + /CD45 − osteoblasts (SNOs) inhibit breast cancer cell proliferation in vitro through the Notch pathway. a MDA GFP cells were seeded onto magnetic-activated cell-sorted (MACS) SNOs and NON-SNOs cultured for 24 h and allowed to attach for 1 h at 37 °C, followed by extensive washing. Number of MDA GFP cells at time = 0 (1 h of adhesion) and after 24–72 h of co-culture. b Number of MDA GFP clones/well after 72 h of culture. c Number of Ki-67-positive MDA GFP cells. d Fluorescence intensity of 4T1 redPKH26 cells seeded onto SNO or NON-SNO monolayers after 24, 48 and 96 h of co-culture. e Number of Ki-67-positive 4T1 cells after 38 h of co-culture. f Notch1–4 expression in MDA GFP cells seeded onto SNOs and NON-SNOs, evaluated after 1 h of adhesion by confocal immunofluorescence. g Number of MDA GFP cells treated with Notch-1–4-specific small interfering RNAs (siRNAs), seeded onto SNOs and evaluated at time 0 (1 h of adhesion) and after 24–72 h, compared with SCR-siRNA-treated MDA GFP cells seeded onto SNOs and NON-SNOs. h MDA GFP cells were treated with the γ-secretase inhibitor dibenzazepine and then seeded onto SNOs. Cell number was evaluated after 1 h (time 0) and again after 24 h. The graph expresses the fold change vs time 0 and results are compared vs dimethyl sulfoxideDMSO-treated MDA GFP seeded onto SNOs and NON-SNOs. i Transcriptional expression of the indicated haematopoietic stem cell (HSC) markers in MACS sorted Notch2 HIGH and Notch2 LOW MDA GFP cell subpopulations. j Flow cytometry of MDA-MB-231 cells after double staining with antibodies for Notch2 and for the HSC markers, CD34, k c-kit and l Notch1. m Morphology, n number and o size of primary mammospheres formed by MACS-sorted Notch2 HIGH and Notch2 LOW MDA LUC subpopulations. p Morphology, q number and r size of secondary mammospheres obtained after trypsinisation and re-plating of the primary mammospheres. Yellow arrowhead, mammosphere. s CD44 and CD24 expression by flow cytometry after double staining of MDA LUC cells with anti-Notch2 antibody. Imaging and data (mean ± SD) represent the results of at least three independent in vitro experiments. Statistical analyses: ( a , d , g ) Non-linear regression fitting and F -test, ( b , c , e , f , h , i , n , q ) two tails’ unpaired t test ( o , r ) Gaussian curve regression fitting and F -test

Article Snippet: Cells and tissue sections were labelled with antibodies against human pan-cytokeratin AE1/AE3, N-cadherin, Notch2 and Ki-67 [Santa Cruz Biotechnology, catalogue numbers, cytokeratin (sc81714), N-cadherin (sc7939), N-cadherin (sc310209), Notch2 (sc5545), Ki-67 (sc7846); Miltenyi Biotech, Notch2-Biotin (130-096-980); Novus, N-Cadherin (48309PE)], dilution 1:100–1:200, either singularly or in combinations as double, triple or quadruple immunofluorescences or stained with haematoxylin and eosin.

Techniques: In Vitro, Cell Culture, Co-Culture Assay, Clone Assay, Fluorescence, Expressing, Immunofluorescence, Flow Cytometry, Double Staining, Imaging

Journal: British Journal of Cancer

Article Title: Notch2 pathway mediates breast cancer cellular dormancy and mobilisation in bone and contributes to haematopoietic stem cell mimicry

doi: 10.1038/s41416-019-0501-y

Figure Lengend Snippet: Notch2 HIGH breast cancer cells. a Size, granularity and b percentage of MDA LUC Notch2 HIGH cell subpopulation after staining with anti-Notch2 antibody, analysed by flow cytometry. c Percentage of Notch2 HIGH MDA LUC cell subpopulation measured after magnetic-activated cell sorting (MACS). d Notch2 HIGH MDA LUC cells analysed in overt osteolytic breast cancer bone metastases. White arrowhead, Notch2 HIGH cell. e Triple immunofluorescence in the tibias of mice injected into the medullary cavity with breast cancer 4T1 cells. f Quadruple immunofluorescence in the tibias of the mouse model of MDA-MB-231 cell dormancy and g in the mouse model of chemotherapy-induced dormancy. B bone. h Graphical scheme of MACS-sorted Notch2 HIGH and Notch2 LOW MDA LUC injection in 5-week-old female immunocompromised CD1 nu/nu mice. i Mouse body weight measured 6 weeks after cancer cell injection. j Micro- computed tomographic analysis of bone volume over total tissue volume (BV/TV) in tibias injected with tumour cells. k Immunofluorescent human cytokeratin area over tissue area, l percentage of metastatic area over total tissue area and m number of multicellular metastases per mm 2 in the histological sections of livers collected from the mice intratibially injected with tumour cells shown in i and j . Imaging and data (mean ± SD) represent the results of at least four mice/group or three independent in vitro experiments. Statistical analyses: two tails’ unpaired t test

Article Snippet: Cells and tissue sections were labelled with antibodies against human pan-cytokeratin AE1/AE3, N-cadherin, Notch2 and Ki-67 [Santa Cruz Biotechnology, catalogue numbers, cytokeratin (sc81714), N-cadherin (sc7939), N-cadherin (sc310209), Notch2 (sc5545), Ki-67 (sc7846); Miltenyi Biotech, Notch2-Biotin (130-096-980); Novus, N-Cadherin (48309PE)], dilution 1:100–1:200, either singularly or in combinations as double, triple or quadruple immunofluorescences or stained with haematoxylin and eosin.

Techniques: Staining, Flow Cytometry, FACS, Immunofluorescence, Injection, Imaging, In Vitro

Journal: British Journal of Cancer

Article Title: Notch2 pathway mediates breast cancer cellular dormancy and mobilisation in bone and contributes to haematopoietic stem cell mimicry

doi: 10.1038/s41416-019-0501-y

Figure Lengend Snippet: Notch2 expression in human primary tumours and correlation with survival. a Analysis of 23 data sets representing transcriptomes from primary breast cancers from 3655 patients and forest plot showing correlation between Notch2 and survival (SurvExpress®). b Kaplan–Meier plots on additional 3951 public transcriptomes from primary breast cancers to correlate Notch2 expression with patient survival in unselected populations of 1976 Notch2 LOW and 1975 Notch2 HIGH samples, c in untreated populations of 506 Notch2 LOW and 504 Notch2 HIGH samples, d in endocrine-treated populations of 938 Notch2 LOW and 935 Notch2 HIGH samples, e in chemotherapy-treated populations of 301 Notch2 LOW and 301 Notch2 HIGH samples, f in oestrogen receptor (ER)-positive of 207 Notch2 LOW and 296 Notch2 HIGH samples and g ER-negative populations of 159 Notch2 LOW and 100 Notch2 HIGH samples, plotted against time, (KMPlot®). h Primary breast cancer histopathologic samples and associated bone metastases were collected and analysed in five unselected patients. Representative immunohistochemistry for Notch2 in mated primary tumours and i associated bone metastasis. B bone. j Graphical representation summarising the findings presented in this article and their interpretation. Statistical analyses: a Z -test, b – g log-rank test

Article Snippet: Cells and tissue sections were labelled with antibodies against human pan-cytokeratin AE1/AE3, N-cadherin, Notch2 and Ki-67 [Santa Cruz Biotechnology, catalogue numbers, cytokeratin (sc81714), N-cadherin (sc7939), N-cadherin (sc310209), Notch2 (sc5545), Ki-67 (sc7846); Miltenyi Biotech, Notch2-Biotin (130-096-980); Novus, N-Cadherin (48309PE)], dilution 1:100–1:200, either singularly or in combinations as double, triple or quadruple immunofluorescences or stained with haematoxylin and eosin.

Techniques: Expressing, Immunohistochemistry