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. 2018 Nov 26;8:17375. doi: 10.1038/s41598-018-35745-1

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© The Author(s) 2018

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-induced up-regulation of sFlt-1 secretion in primary cytotrophoblasts. Thawed primary cytotrophoblasts were cultured for 16 h and then incubated for 24 h under normoxic or hypoxic conditions. Conditioned media were collected and analyzed for secretion of sFlt-1 proteins by Western blot analysis. (A) The mRNA expression of all Flt-1 transcripts (FLT-1) in cytotrophoblasts was assessed by quantitative real-time PCR analysis using β-actin mRNA as a reference. Results are represented as a fold change relative to cells under normoxic conditions. (B) The mRNA expression levels of three Flt-1 splice variants (tmFlt-1, sFlt-1 i13, and sFlt-1 e15a) in cytotrophoblasts. Results are represented as a ratio relative to the expression of β-actin mRNA. (C) Secreted sFlt-1 proteins in conditioned media were concentrated by heparin-sepharose beads and then subjected to Western blot analysis with anti-human Flt-1 N-terminal antibody. Uncropped image of Western blot is presented in Supplementary Fig. S5. All values are represented as the means ± SD (n = 3). Asterisks indicate a significant difference (P < 0.05).