Proteomics 2015, 15, 2945–2952 2945 DOI 10.1002/pmic.201500067 RESEARCH ARTICLE Identification of proteins involved in inhibition of spheroid formation under microgravity Stefan Riwaldt 1 , Jessica Pietsch 1 , Albert Sickmann 2 , Johann Bauer 3 , Markus Braun 4 , Juergen Segerer 5 , Achim Schwarzw¨ alder 5 , Ganna Aleshcheva 1 , Thomas J Corydon 6 , Manfred Infanger 1 and Daniela Grimm 6 1 Clinic for Plastic, Aesthetic and Hand Surgery, Otto-von-Guericke-University-Magdeburg, Magdeburg, Germany 2 Leibniz-Institut f ¨ ur Analytische Wissenschaften –ISAS- e.V., Dortmund, Germany 3 Max-Planck Institute for Biochemistry, Martinsried, Germany 4 Institute for Molecular Physiology and Biotechnology of Plants (IMBIO), Gravitational Biology Group, University of Bonn, Bonn, Germany 5 Airbus, Defense and Space (ADS), Immenstaad, Germany 6 Department of Biomedicine, Aarhus University, Aarhus, Denmark Received: February 13, 2015 Revised: March 27, 2015 Accepted: April 24, 2015 Many types of cells transit in vitro from a two- to a three-dimensional growth, when they are exposed to microgravity. The underlying mechanisms are not yet understood. Hence, we inves- tigated the impact of microgravity on protein content and growth behavior. For this purpose, the human thyroid cancer cells FTC-133 were seeded either in recently developed cell contain- ers that can endure enhanced physical forces and perform media changes and cell harvesting automatically or in T-25 culture flasks. All cells were cultured for five days at 1g. Afterwards, a part of the cell containers were flown to the International Space Station, while another part was kept on the ground. T-25 flasks were mounted on and next to a Random Positioning Machine. The cells were cultured for 12 days under the various conditions, before they were fixed with RNAlater. All fixed cultures showed monolayers, but three-dimensional aggregates were not detected. In a subsequent protein analysis, 180 proteins were identified by mass spectrometry. These proteins did not indicate significant differences between cells exposed to microgravity and their 1g controls. However, they suggest that an enhanced production of proteins related to the extracellular matrix could detain the cells from spheroid formation, while profilin-1 is phosphorylated. Keywords: Cell biology / Microgravity / Profilin-1 / Random Positioning Machine / Spaceflight / Thyroid cancer cells  Additional supporting information may be found in the online version of this article at the publisher’s web-site Correspondence: Dr. Johann Bauer, Max-Planck-Institute for Bio- chemistry, Am Klopferspitz 18a, D-82152 Martinsried, Germany E-mail: jbauer@biochem.mpg.de Fax: +49 89 1417931 Abbreviations: ECM, extracellular matrix; FFM, cell container suitable for spaceflights flown to the ISS; FM, cell container suit- able for spaceflights; GFM, cell container suitable for spaceflights remaining on the ground; GuHCl, guanidin hydrochlorid; ISS, In- ternational Space Station; RPM, Random Positioning Machine; TEAB, tetraethylamonium bromide 1 Introduction Research on numerous types of human cells exposed to weightlessness has been conducted in recent years [1]. The scientists wanted to understand the health problems, which Space travelers often face, and aimed also to learn about ge- netic changes and cancer development [2, 3]. An important aspect of these studies is the comparison of protein contents of cells grown under normal 1g and of cells exposed to weight- lessness [4]. As spaceflights are rare and extremely expensive, Colour Online: See the article online to view Figs. 1, 2 and 4 in colour. C  2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com