Journal: Brain : a journal of neurology

Article Title: Pilocytic astrocytoma of the optic pathway: a tumour deriving from radial glia cells with a specific gene signature.

doi: 10.1093/brain/awp048

Figure Lengend Snippet: Figure 2 (A and B) Microarrays analysis was confirmed by RT-Q-PCR and immunohistochemistry experiments. (A) LHX2 (left, P = 0.045) and SIX6 (right, P = 0.008) mRNA expression measured by RT-Q-PCR analysis demonstrate higher level in H/C PA versus cerebellar PA. (B) PTEN expression by immunohistochemistry was higher in H/C PA than in cerebellar PA. Scale bar: 150.7 mm. This difference was statistically significant. (C) Quantification of CDK5, LHX2, NOTCH2 and SIX6 in H/C PA and cerebellar PA. The percentage of immunolabelled surface with anti-CDK5, NOTCH2 and SIX6 antibodies, in H/C PA was statistically different from that observed in cerebellar PA. The percentage of surface labelled for LHX2 in H/C PA was higher than in cerebellar PA but does not reach statistical significance.

Article Snippet: After steam-heat-induced antigen retrival, 5 mm sections of formalin-fixed paraffin-embedded samples were tested for the presence of PTEN (1/1000, mouse IgG, Santa Cruz Biotechnology, CA, USA), CDK5 (1/1000, rabbit IgG, Santa Cruz Biotechnology), SIX6 (1/1000, Goat IgG, Santa Cruz Biotechnology), NOTCH2 (1/500, rabbit IgG, Santa Cruz Biotechnology) and LHX2 (1/100, rabbit IgG, Chemicon International, Saint-Quentin en Yvelines, France).

Techniques: Immunohistochemistry, Expressing

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