notch1 blocking  (MedChemExpress)

Journal: Scientific Reports

Article Title: Macrophage-dependent tumor cell transendothelial migration is mediated by Notch1/Mena INV -initiated invadopodium formation

doi: 10.1038/srep37874

Figure Lengend Snippet: ( A ) Immunofluorescence of MDA-MB-231 tumor cell in contact with BAC1.2F5 macrophage plated on 405-gelatin. Tumor cells were stained for Tks5 (purple) and cortactin (green) to identify invadopodium cores (insert is zoom of mature invadopodia). Position of the macrophage is indicated by the arrow. ( B ) Quantitation of the number of mature invadopodia per cell in MDA-MB-231 tumor cells plated alone or with BAC1.2F5 macrophages. Cells were treated with vehicle or DAPT γ- secretase inhibitor. ( C ) Quantitation of the number of mature invadopodia per cell in MDA-MB-231 tumor cells treated with control or Notch1 siRNA plated with BAC1.2F5 macrophages. ( D ) Quantitation of the area of matrix degradation in control and Notch1 siRNA treated MDA-MB-231 cells plated with BAC1.2F5 macrophages. ( E ) Western blot of MDA-MB-231 cells treated with control and Notch1 siRNA demonstrating knockdown efficiency. mDia1 (mammalian homolog of Drosophila diaphanous) was used as a loading control. * P < 0.05, ** P < 0.005, *** P < 0.0005.

Article Snippet: Notch1 function blocking antibody (R&D Systems) was used in vitro at 5 μg/mL and in vivo via intraperitoneal injection at 1 mg/kg.

Techniques: Immunofluorescence, Staining, Quantitation Assay, Control, Western Blot, Knockdown

Journal: Scientific Reports

Article Title: Macrophage-dependent tumor cell transendothelial migration is mediated by Notch1/Mena INV -initiated invadopodium formation

doi: 10.1038/srep37874

Figure Lengend Snippet: ( A ) qRT-PCR of invadopodia pathway components in MDA-MB-231 cells plated alone or with BAC1.2F5 macrophages (Mϕ). ( B ) qRT-PCR of Mena INV in MDA-MB-231 cells plated in transwells alone, co-cultured in top well of transwells in contact with BAC1.2F5 macrophages (Mϕ) or plated on opposite side of transwells (no contact). ( C ) qRT-PCR of Mena INV in MDA-MB-231 cells treated with control or Notch1 siRNA plated alone or with BAC1.2F5 macrophages. ( D ) qRT-PCR of Mena INV in MDA-MB-231 cells treated with vehicle or DAPT γ- secretase inhibitor plated alone or with BAC1.2F5 macrophages. * P < 0.05, ** P < 0.005, *** P < 0.0005.

Article Snippet: Notch1 function blocking antibody (R&D Systems) was used in vitro at 5 μg/mL and in vivo via intraperitoneal injection at 1 mg/kg.

Techniques: Quantitative RT-PCR, Cell Culture, Control

Journal: Scientific Reports

Article Title: Macrophage-dependent tumor cell transendothelial migration is mediated by Notch1/Mena INV -initiated invadopodium formation

doi: 10.1038/srep37874

Figure Lengend Snippet: ( A ) Quantitation of the relative mRNA expression in MDA-MB-231 cells treated with control, panMena or MenaI NV siRNA in the absence of macrophages. ( B ) Quantitation of the relative number of invadopodia in MDA-MB-231 cells treated with control, panMena or Mena INV siRNA in the presence of macrophages. ( C ) Representative apical z-section of intravasation-directed transendothelial migration (iTEM) assay of tumor cells (green) and macrophages (blue, white arrows). Tumor cells were treated with control or Notch1 siRNA. Endothelial HUVEC cells were stained for ZO-1 (red). Shown is an “en face” view of the apicial side of the transwell, therefore the green-labeled tumor cells have crossed the endothelial monolayer and are on the ‘bottom’ or apical side of the transwell positioning them above the endothelial cells from this vantage point. ( D ) Quantitation of intravasation-directed is defined as subluminal to luminal transendothelial migration (iTEM) activity of MDA-MB-231 cells plated on the endothelium either alone or with BAC1.2F5 macrophages. Tumor cells were treated with control or Notch1 siRNA. ( E ) Quantitation of iTEM activity of MDA-MB-231 cells plated alone or with BAC1.2F5 macrophages with vehicle or DAPT treatment. * P < 0.05, ** P < 0.005, *** P < 0.0005.

Article Snippet: Notch1 function blocking antibody (R&D Systems) was used in vitro at 5 μg/mL and in vivo via intraperitoneal injection at 1 mg/kg.

Techniques: Quantitation Assay, Expressing, Control, Migration, Staining, Labeling, Activity Assay

Journal: Scientific Reports

Article Title: Macrophage-dependent tumor cell transendothelial migration is mediated by Notch1/Mena INV -initiated invadopodium formation

doi: 10.1038/srep37874

Figure Lengend Snippet: ( A ) Immunofluorescence images of MTLn3 tumor cells plated in the presence or absence of BAC1.2F5 macrophages (Mϕ) and treated with control IgG or Notch1 blocking IgG. Macrophages were labeled with cell tracker red and cells were stained for cortactin and Tks5. ( B ) Quantitation of the relative number of invadopodia per cell in MTLn3 cells plated alone or with Mϕ and treated with control IgG or Notch1 blocking IgG. ( C ) Dendra2-MTLn3 xenograph mammary tumors in SCID nice were imaged using a mammary imaging window. Regions in vascularized areas were photo-converted in mice treated with control IgG or Notch1 blocking IgG. Photo-converted cells were tracked at 0 and 24 hours. ( D ) Quantitation of the percent of red photo-converted cells remaining in the photo-converted region at 24 hours measures amount of intravasation. ( E ) Quantitation of the circulating tumor cells from SCID mice with MTLn3 xenographs. Mice were treated with control IgG or Notch1 blocking IgG. * P < 0.05, ** P < 0.005, *** P < 0.0005.

Article Snippet: Notch1 function blocking antibody (R&D Systems) was used in vitro at 5 μg/mL and in vivo via intraperitoneal injection at 1 mg/kg.

Techniques: Immunofluorescence, Control, Blocking Assay, Labeling, Staining, Quantitation Assay, Imaging