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. 2019 Dec 20;98(2):193–207. doi: 10.1007/s00109-019-01866-x

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© The Author(s) 2019

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — Platelet-derived factors do not affect cAMP/CREB signaling, but activated Smad3 in BeWo differentiation. Levels of intracellular cAMP (a) and phosphorylated CREB (b) were analyzed by ELISA in forskolin-stimulated (20 μM) and vehicle control (DMSO, 0.1% v/v) BeWo cells in the presence and absence of pHPL at indicated time points. Moreover, phosphorylated CREB (pCREB) was analyzed by immunoblot (c) and band densitometry (d) after 1 h treatment. Expression of CREB target gene NR4A2 (e) was analyzed in BeWo cells after forskolin induction in the presence and absence of pHPL after 1 h. Phosphorylated Smad3 (pSmad3) was analyzed by immunoblot (f) and band densitometry (g) after 1 h treatment. Expression of Smad3 target gene SERPINE1 (h) was analyzed in BeWo cells after 48 h. Data in bar graphs a and b are presented as means ± SEM from five independent experiments using different cell passages and were tested for differences using two-way analysis of variance followed by Tukey’s multiple comparisons test. Western blots are representative for three (c) and eight (f) different experiments. For band densitometry (d, g), controls were set to one and data were tested using one sample t test. Data in e and h were analyzed using one-way analysis of variance and Tukey’s multiple comparisons test. *p ≤ 0.05, **p ≤ 0.01, ***p < 0.001