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. 2011 Jul 1;6(7):e20444. doi: 10.1371/journal.pone.0020444

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Grandclement et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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A, HT29^ctrl, HT29^NRP2, Colo320^siRNA-NRP2 and Colo320^siRNA-ctrl were analyzed for expression of epithelial and mesenchymal markers (respectively E-Cadherin and vimentin) by western-blotting (whole cell lysate). β-actin was used as a control of protein loading. The presence of NRP2 decreases E-cadherin expression and induces vimentin in HT29 colorectal cancer cells. Specific disruption of NRP2 using siRNA down-regulated vimentin in Colo320 cancer cells. Blotted proteins have been quantified with the BIO-1D advanced software and reported to the β-actin level. B, Frozen sections of the isolated tumors from HT29^ctrl and HT29^NRP2 xenografts were subjected to immunohistochemical staining with anti-cytokeratin-20, anti-Ecadherin antibodies and HES staining (magnification x100). HES (Hematoxiline Eosine Safran) staining was used to assess morphology. HT29^NRP2 invade smooth muscle (sm = smooth muscle) as indicated by arrows unlike HT29^ctrl which showed a local invasion. HT29^NRP2 xenografts lacked epithelial markers such as cytokeratin-20 and E-cadherin. C, Pancreatic and renal cancer cell lines were analyzed for NRP2, E-cadherin and vimentin expression by western-blotting. β-actin was used as a control of protein loading. The presence of NRP2 is positively correlated with mesenchymal marker vimentin and inversely correlated with epithelial marker E-cadherin. D, Transcriptional factors Snail1 and Twist1 were analyzed in HT29^ctrl, HT29^NRP2, Colo320^siRNA-NRP2 and Colo320^siRNA-ctrl whole cell lysates by western-blotting. NRP2 expressing cells (Colo320^siRNA-ctrl and HT29^NRP2) have increased levels of Snail1 and Twist1 compared to NRP2 lacking cells (HT29^ctrl and Colo320^siRNA-NRP2). β-actin was used as a control of protein loading. Blotted proteins have been quantified with the BIO-1D advanced software and reported to the β-actin level. E, Evaluation of NRP2, Snail, Twist1, Gli1 and TGFβ1 expression by QRT-PCR analysis in Colo320^siRNA-ctrl and Colo320^siRNA-NRP2. Decreased NRP2 expression is indeed associated with a down-regulation of transcriptional regulators Snail, Gli1 and twist1 and with a decreased TGF-β1 production. F, Confocal microscopy analysis of localization of β-catenin. Cells (HT29^ctrl and HT29^NRP2) were stained with DAPI (blue) for nuclear staining and also with anti-β-catenin antibody (green). Whereas β-catenin (green) is localized at the membrane in HT29^ctrl cells, HT29^NRP2 cells show a nuclear localization of this transcriptional activator.

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