Journal: International Journal of Oncology

Article Title: Regulation of gliomagenesis and stemness through acid sensor ASIC1a

doi: 10.3892/ijo.2021.5262

Figure Lengend Snippet: Notch signaling is negatively associated with ASIC1a in glioblastoma multiforme. (A-C) The glioma cells were transfected with shASIC1a and control followed by assaying protein expression of ASIC1a, active form of Notch1, Notch2, Notch3, Notch4, and Notch target survivin, as well as GSC markers CD133 and ALDH1 by western blotting in (A) A172, (B) U87MG, and (C) PDX-L12 cells. (D-F) Subsequently, the glioma cells (D) A172, (E) U87MG and (F) PDX-L12 were transfected with ASIC1a-FLAG followed by assaying protein expression of ASIC1a, active form of Notch1, Notch2, Notch3, Notch4 and Notch target survivin, as well as GSC markers CD133 and ALDH1 by western blotting. * P<0.05 and ** P<0.01. ASIC1a, acid-sensing ion channel 1a; sh, short hairpin; GSC, glioblastoma stem cells; ALDH1, aldehyde dehydrogenase 1.

Article Snippet: The following primary antibodies were used in the present study: Rabbit monoclonal anti-ASIC1a antibody (cat. no. 35-156465) was purchased from American Research Products, Inc. Rabbit polyclonal anti-Notch4 antibody (cat. no. 07-189) and anti-β-actin antibody (product no. A3854) were purchased from Sigma-Aldrich; Merck KGaA.

Techniques: Transfection, Control, Expressing, Western Blot

Journal: BMC Genomics

Article Title: Characterization of gene expression profiles for different types of mast cells pooled from mouse stomach subregions by an RNA amplification method

doi: 10.1186/1471-2164-10-35

Figure Lengend Snippet: Summary of genes examined by real-time PCR analysis.

Article Snippet: For tissue staining, frozen sections were fixed in 4% formaldehyde and incubated with a rabbit anti-Notch4 antibody (1:20, Santa Cruz Biotechnology, Santa Cruz, CA) or a rabbit anti-Ptgr1 antibody (1:20) which was a kind gift from Prof. Takao Shimizu (University of Tokyo) [ ].

Techniques: Real-time Polymerase Chain Reaction

Journal: BMC Genomics

Article Title: Characterization of gene expression profiles for different types of mast cells pooled from mouse stomach subregions by an RNA amplification method

doi: 10.1186/1471-2164-10-35

Figure Lengend Snippet: Immunohistochemical analysis of Notch4 and Ptgr1 in sMCs and mMCs in stomach tissue . ( a ) Stomach submucosa (sMCs; left panels ), stomach mucosa (mMCs; middle panels ) and skin (skin MCs; right panels ) sections were stained with an anti-Notch4 antibody ( lower panels ) and with toluidine blue ( upper panels ). sMCs stained with the anti-Notch4 antibody in the gastric submucosa and skin dermis are indicated by arrows. No staining was observed in mMCs ( arrowheads ) localized in the gastric mucosa. sMCs and mMCs were metachromatically stained with toluidine blue. ( b ) Stomach submucosa (sMCs; left panels ) and stomach mucosa (mMCs; right panels ) sections were stained with an anti-Ptgr1 antibody ( lower panels ) and with toluidine blue ( upper panels ). No staining with the anti-Ptgr1 antibody was found in the sMCs ( arrow ). Small signals were observed in the mMCs ( arrowheads ). sMCs and mMCs were metachromatically stained with toluidine blue. Bars , 25 μm (a, b).

Article Snippet: For tissue staining, frozen sections were fixed in 4% formaldehyde and incubated with a rabbit anti-Notch4 antibody (1:20, Santa Cruz Biotechnology, Santa Cruz, CA) or a rabbit anti-Ptgr1 antibody (1:20) which was a kind gift from Prof. Takao Shimizu (University of Tokyo) [ ].

Techniques: Immunohistochemical staining, Staining

Journal: BMC Genomics

Article Title: Characterization of gene expression profiles for different types of mast cells pooled from mouse stomach subregions by an RNA amplification method

doi: 10.1186/1471-2164-10-35

Figure Lengend Snippet: List of primers used for real-time PCR analysis.

Article Snippet: For tissue staining, frozen sections were fixed in 4% formaldehyde and incubated with a rabbit anti-Notch4 antibody (1:20, Santa Cruz Biotechnology, Santa Cruz, CA) or a rabbit anti-Ptgr1 antibody (1:20) which was a kind gift from Prof. Takao Shimizu (University of Tokyo) [ ].

Techniques: Real-time Polymerase Chain Reaction

Journal: Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine

Article Title: EGFL7 participates in regulating biological behavior of growth hormone-secreting pituitary adenomas via Notch2/DLL3 signaling pathway.

doi: 10.1177/1010428317706203

Figure Lengend Snippet: Figure 3. EGFL7 protein is highly expressed in invasive GHPA tissue. (a) Representative western blots of EGFL7 and Notch2 in invasive and non-invasive GHPA. Blots were reprobed with anti-GAPGH antibody to ensure equal loading. (b) Quantitative analysis of western blots showed that EGFL7 and Notch2 expression was markedly higher in invasive GHPA than in non-invasive GHPA. (c) Mean H-scores°±°SD of EGFL7 staining of tissue microarrays. *p < 0.05 versus normal pituitary; **p < 0.001 versus normal pituitary; #p < 0.05 versus non-invasive pituitary adenoma. (d) Representative images of EGFL7 staining of a tissue microarray showing that cytoplasmic EGFL7 staining was significantly higher in invasive GHPA than in non-invasive GHPA and normal pituitary tissue. EGFL7- positive endothelial cells can be observed in immunostained slides (red arrows) (scale bar: 60 µm).

Article Snippet: Membranes were then blocked in Tris-buffered saline (TBS) buffer containing 5% degreased milk for 1 h at room temperature and incubated with primary antibody overnight at 4°C, rabbit polyclonal anti-EGFL7 antibody (1:500, H-90 sc-66874; Santa Cruz Biotechnology; 1:500, 19291-1-AP; Proteintech, Chicago, IL, USA), mouse monoclonal anti-EGFL7 antibody (1:2000, 2H2 sc-101349; Santa Cruz Biotechnology, California, USA), rabbit polyclonal anti-Notch1 (1:2000, ab27526; Abcam, Cambridge, USA), rabbit monoclonal anti-Notch1 (1:5000, ab194123; Abcam, Cambridge, USA), rabbit polyclonal anti-Notch2 (1:2000, ab8926; Abcam, Cambridge, USA), rabbit monoclonal anti-Notch4 (1:2000, ab184742; Abcam, Cambridge, USA), rabbit polyclonal anti-DLL3 (1:2000, ab10554; Abcam, Cambridge, USA), rabbit polyclonal antiDLL3 (1:2000, ab63707; Abcam, Cambridge, USA), rabbit polyclonal anti-DLL4 (1:2000, ab7280; Abcam, Cambridge, USA), and GAPDH (1:8000; Sigma, St Louis, MO, USA) followed by secondary antibodies tagged with horseradish peroxidase (Abcam, Cambridge, USA).

Techniques: Western Blot, Expressing, Staining, Microarray

Journal: Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine

Article Title: EGFL7 participates in regulating biological behavior of growth hormone-secreting pituitary adenomas via Notch2/DLL3 signaling pathway.

doi: 10.1177/1010428317706203

Figure Lengend Snippet: Figure 4. Lentivirus-mediated knockdown of endogenous EGFL7 expression and differential expression of Notch pathway. (a) Representative western blots of downregulated EGFL7 expression by RNAi and differential expression of Notch pathway. (b) and (c) Quantitative analysis of western blots showed GH3 cells transfected with sh-C or sh-B downregulated EGFL7 expression more efficiently. Knockdown of EGFL7 induced low expression of Notch2/DLL3. However, there are no significant alteration in Notch1, 4 and DLL1, 4. *p < 0.05 versus control and non-silence shRNA. (d) qRT–PCR analysis demonstrated a significantly reduction in EGFL7 mRNA expression after transfected with sh-C and sh-B. *p < 0.05 versus control and non-silence shRNA.

Article Snippet: Membranes were then blocked in Tris-buffered saline (TBS) buffer containing 5% degreased milk for 1 h at room temperature and incubated with primary antibody overnight at 4°C, rabbit polyclonal anti-EGFL7 antibody (1:500, H-90 sc-66874; Santa Cruz Biotechnology; 1:500, 19291-1-AP; Proteintech, Chicago, IL, USA), mouse monoclonal anti-EGFL7 antibody (1:2000, 2H2 sc-101349; Santa Cruz Biotechnology, California, USA), rabbit polyclonal anti-Notch1 (1:2000, ab27526; Abcam, Cambridge, USA), rabbit monoclonal anti-Notch1 (1:5000, ab194123; Abcam, Cambridge, USA), rabbit polyclonal anti-Notch2 (1:2000, ab8926; Abcam, Cambridge, USA), rabbit monoclonal anti-Notch4 (1:2000, ab184742; Abcam, Cambridge, USA), rabbit polyclonal anti-DLL3 (1:2000, ab10554; Abcam, Cambridge, USA), rabbit polyclonal antiDLL3 (1:2000, ab63707; Abcam, Cambridge, USA), rabbit polyclonal anti-DLL4 (1:2000, ab7280; Abcam, Cambridge, USA), and GAPDH (1:8000; Sigma, St Louis, MO, USA) followed by secondary antibodies tagged with horseradish peroxidase (Abcam, Cambridge, USA).

Techniques: Knockdown, Expressing, Quantitative Proteomics, Western Blot, Transfection, Control, shRNA, Quantitative RT-PCR

Journal: Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine

Article Title: EGFL7 participates in regulating biological behavior of growth hormone-secreting pituitary adenomas via Notch2/DLL3 signaling pathway.

doi: 10.1177/1010428317706203

Figure Lengend Snippet: Figure 5. EGFL7 downregulation suppressed invasion and proliferation of GH3 cells. (a) Representative transwell invasion assays of GH3 cells were performed after knockdown of EGFL7. (b) Quantitative analysis indicated the invasion of GH3 cells decreased by about threefold with sh-C transfection and decreased twofold with sh-B transfectioncompared to transfected with non-silence shRNA. (c) MTT assay revealed that knockdown of EGFL7 suppresses GH3 cell proliferation rate.

Article Snippet: Membranes were then blocked in Tris-buffered saline (TBS) buffer containing 5% degreased milk for 1 h at room temperature and incubated with primary antibody overnight at 4°C, rabbit polyclonal anti-EGFL7 antibody (1:500, H-90 sc-66874; Santa Cruz Biotechnology; 1:500, 19291-1-AP; Proteintech, Chicago, IL, USA), mouse monoclonal anti-EGFL7 antibody (1:2000, 2H2 sc-101349; Santa Cruz Biotechnology, California, USA), rabbit polyclonal anti-Notch1 (1:2000, ab27526; Abcam, Cambridge, USA), rabbit monoclonal anti-Notch1 (1:5000, ab194123; Abcam, Cambridge, USA), rabbit polyclonal anti-Notch2 (1:2000, ab8926; Abcam, Cambridge, USA), rabbit monoclonal anti-Notch4 (1:2000, ab184742; Abcam, Cambridge, USA), rabbit polyclonal anti-DLL3 (1:2000, ab10554; Abcam, Cambridge, USA), rabbit polyclonal antiDLL3 (1:2000, ab63707; Abcam, Cambridge, USA), rabbit polyclonal anti-DLL4 (1:2000, ab7280; Abcam, Cambridge, USA), and GAPDH (1:8000; Sigma, St Louis, MO, USA) followed by secondary antibodies tagged with horseradish peroxidase (Abcam, Cambridge, USA).

Techniques: Knockdown, Transfection, shRNA, MTT Assay