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BioMed Research International
Volume 2014, Article ID 413708, 11 pages
http://dx.doi.org/10.1155/2014/413708
Research Article

The Influence of Simulated Microgravity on Purinergic Signaling Is Different between Individual Culture and Endothelial and Smooth Muscle Cell Coculture

1Department of Natural Sciences, Bonn-Rhine-Sieg University of Applied Sciences, 53359 Rheinbach, Germany
2Institute of Pharmacology and Medical Chemistry, University of Dusseldorf, 40225 Dusseldorf, Germany
3Institute of Aerospace Medicine, German Aerospace Center, 51147 Cologne, Germany

Received 25 April 2014; Revised 30 June 2014; Accepted 23 July 2014; Published 28 August 2014

Academic Editor: Monica Monici

Copyright © 2014 Yu Zhang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Supplementary Material

The table 1 is showed for the number of endothelial cell numbers culturing under DMEM, SMC CM+1g and SMC CM+MG conditions for 24h, 48h respectively. The figure 5(a) is presented as one example (Donor 1) of three individual cows.

The table 2 is showed for the number of migrated endothelial cell numbers culturing under DMEM, SMC CM+1g and SMC CM+MG conditions for 24h. The figure 5(b) is presented as one example (Donor 1) of three individual cows.

The table 3 is showed for the number of smooth muscle cell numbers culturing under DMEM, EC CM+1g and EC CM+MG conditions. The figure 6(a) is presented as one example (Donor 1) of three individual cows.

The table 4 is showed for the number of migrated smooth muscle cell numbers culturing under DMEM, EC CM+1g and EC CM+MG conditions. The figure 6(b) is presented as one example (Donor 1) of three individual cows.

The amplification conditions for each specific product were conducted as followed: the initial denaturation was performed at 94°C for 3 min and cyclic denaturation was run for 30 seconds. The cyclic annealing step was performed for 30 seconds with the respective annealing temperatures (see table 5). The target gene was elongated at 72°C for 45 seconds. The cyclic amplification was repeated with different numbers (see table 5). A final extension of 3 min at 72°C was set with subsequent cooling down to 4°C. GAPDH served as the housekeeping gene controls, which were adjusted to equal levels prior to the comparison of genes of interest. The RT-PCR product was evaluated with 1% agarose gel electrophoresis. The images of gels were taken with a Bio-Rad Chemidoc machine.

The cell line HMEC-1 and C2 were used as positive controls to confirm the EC and SMC specific markers, respectively. Since no single cell type can expresses all P2 receptor subtypes, different positive controls were used here: HMEC-1 for P2X3, P2X4, P2X5, P2X7, P2Y1, P2Y2, P2Y4, P2Y11, P2Y12; MG-63 for P2X6, P2Y6 and U-87 MG for P2X1, P2X2, P2Y13, P2Y14. The GAPDH of the positive control is only given for HMEC-1 as representative for all three positive controls.

  1. Supplementary Material