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. 2020 Jun 25;10:10345. doi: 10.1038/s41598-020-67217-w

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© The Author(s) 2020

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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Hypoxia induces cell surface translocation of nucleolin under hypoxic conditions. (a) PASMCs were lysed following the exposure to normoxia or hypoxia (FiO₂ 0.03) for 1 h. Nucleolin (NCL), low density lipoprotein related protein 1 (LRP1), integrin β1, epidermal growth factor receptor (EGFR), and β-tubulin expression levels were evaluated. Representative images of western blot, and their quantification (n = 4). Full-length blots are presented in Supplementary Figure 7. (b) Nucleolin (NCL), LRP1, integrin β1, lamin B, and Na/K-ATPase expression in nuclear or membrane fractions of pulmonary arterial smooth muscle cells (PASMCs) after their exposure to hypoxia or normoxia, and their quantification (n = 6). Full-length blots are presented in Supplementary Figure 8. (c) The distribution of green fluorescent protein (GFP)-tagged nucleolin was observed under hypoxia, using time-lapse microscopy. (d) Confocal laser microscopy image of GFP-tagged nucleolin and Na/K-ATPase, under hypoxic conditions. (e) PASMCs were treated with biotinylated midkine (MK) or vehicle, and exposed to normoxia or hypoxia, and midkine-nucleolin binding was analyzed. Full-length blots are presented in Supplementary Figure 9. (f) NCL-EGFR interactions in PASMCs treated with MK or vehicle, and exposed to hypoxia. Strep-HRP, horseradish peroxidase conjugate streptavidin. Full-length blots are presented in Supplementary Figure 10.