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. 2017 Nov 21;7:15923. doi: 10.1038/s41598-017-16184-w

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Comparative analyses of CD157 proteoforms in HeLa cell adhesion to ECM components, wound healing and enzymatic activities. (a) Histograms of adhesion assay with HeLa/pBST1-001 (blue bars) and HeLa/pBST1-002 (orange bars) transfectants show increase in adhesion to indicated substrates with respect to mock control (white bar). Cells were plated for 10 min at 37 °C onto fibronectin, collagen I or vitronectin. Results are expressed as fold-increase of cell adhesion in BST1-001- or BST1-002-transfected HeLa cells compared to mock-transfected cells, and represent the mean value ± s.e.m. of three experiments performed in sextuplicate. (b) Scratch-wound assay comparing the ability to migrate of HeLa/pBST1-001 or pBST1-002. Results represent the distance in μm travelled by cells in 24 h determined as difference between the width of the wound at time 0 and at 24 h, and are expressed as mean ± s.e.m. of three independent experiments performed in triplicate. ****p < 0.0001; ***p < 0.001; **p < 0.01; ns, not significant; ANOVA with Dunnett’s multiple comparison test; (c) HeLa transfectants were incubated for 30 min without (left panel) or with FCS (right panel) then lysed. Total lysates (30 μg/lane) were resolved by 10% SDS-PAGE, immunoblotted and probed with the indicated antibodies. The numbers below each sample correspond to the relative protein density measured in HeLa/pBST1-001 or pBST1-002 compared with HeLa/mock and appropriate total protein. One representative experiment is shown (n = 3). Full-length blots are presented in Supplementary Figure 4 (lower panel). (d) Fluorimetric determination of ectoNADase activity or (e) ectoGDP-ribosyl cyclase activity in culture supernatants from HeLa/pBST1-001 (blue squares), HeLa/pBST1-002 (orange triangles) and HeLa mock transfectants (black circles) incubated with ε-NAD⁺ or NGD⁺, respectively. Raji Burkitt’s lymphoma cell line (CD38-positive, CD157-negative) was used as positive control. Accumulation of fluorescent products ε-ADPR or cGDPR was measured over time and is represented in arbitrary units (AU) of fluorescence intensity measured at 430 nm. Results are the mean ± s.d. of two independent experiments performed in triplicate.