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. 2017 Jun 15;8:15838. doi: 10.1038/ncomms15838

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Copyright © 2017, The Author(s)

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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(a–e) Representative confocal images of in vitro differentiated MKs reveal polarized DMS in mature wt MKs (a), defective polarization in Gp1ba^−/−;Tg (Gp1ba-Tg) (b) and Cdc42^−/− MKs (c), increased polarization in RhoA^−/− MKs (d) and defective polarization of RhoA^−/−MKs after treatment with GPIbα-blocking monovalent Fab fragments, p0p/B-Fab (e). F-actin is stained by phalloidin (red); GPIbβ (a–d) or GPIX (e), green; DAPI, blue. 3D surface rendering of respective z-stacks (Imaris software; right panel). n=5 biological replicates. Scale bar: 10 μm. (f) Quantification of DMS polarization shows reduced MK polarization in Gp1ba-Tg (light grey) and Cdc42^−/− (white) MKs compared to the wt (black) (n=5 biological replicates). (g) MK polarization is increased in RhoA^−/− MKs (light grey) compared to the untreated wt (black) and wt MKs treated with p0p/B-Fab fragments (dark grey). Treatment of RhoA^−/− MKs with p0p/B-Fab fragments (white) reverts the hyperpolarization. n=5 biological replicates (wt and RhoA^−/−) and 2 biological replicates (wt+p0p/B-Fab and RhoA^−/−+p0p/B-Fab). Bar graphs represent mean±s.d. Two-way ANOVA with Bonferroni correction for multiple comparisons; ***P<0.001.