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Stem Cells International
Volume 2016, Article ID 2868323, 10 pages
Research Article

Role of Nitric Oxide, Nitric Oxide Synthase, Soluble Guanylyl Cyclase, and cGMP-Dependent Protein Kinase I in Mouse Stem Cell Cardiac Development

1Department of Neurofarba, Pharmacology and Toxicology Unit, University of Florence, Florence, Italy
2Interuniversity Center for Molecular Medicine and Applied Biophysics, Florence, Italy

Received 9 August 2016; Accepted 26 September 2016

Academic Editor: Michael Lichtenauer

Copyright © 2016 Valentina Spinelli 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

Table 1S: Isolation of total RNA, Reverse Transcription (RT) and Real Time PCR (Q-PCR). Samples of mEBs were collected at different time-points of differentiation for molecular assessment by RNA extraction, reverse transcription, and gene expression analysis using quantitative real-time PCR (Q-PCR) and real-time quantitative PCR (Q-PCR) was performed. The amplicon context sequences of the primers were listed in Table 1S.

Figure 1S: Gene expression profile of pluripotency (mNanog), early mesodermic (mBrachyury) and mature cardiac marker (mMef2c) genes in mESCs and during cardiac differentiation. Gene expression analysis of pluripotency (mNanog), early mesodermic (mBrachyury) and mature cardiac marker (mMef2c) was performed in undifferentiated mESCs and during cardiac differentiation. During the differention, the pluripotency transcription factor mNanog steadily dropped; mMef2c was progressively up-regulated from day 0 to day 8 and then declined, following a bell-shaped curve. The mesodermal marker mBrachyury, showed a transient expression pattern peaking at day 2.

Figure 2S: PGK-I protein expression levels of samples used to quantify serine/threonine phosphorylation by PKG-I. PGK-I protein expression levels were detected in each experimental sample used to quantify serine/threonine phosphorylation by PKG-I. As shown in figure 2S, western blot analysis confirmed that PKG-I expression level was very similar in each sample, ruling out the possibility that differences in phosphorylation could be dependent on PKG-I expression variability.

Figure 3S: Lactate hydrogenase activity. LDH activity was 3,01± 0,604 mU/ml in control conditions at day 4. This value remained similar, independently of the maturation day. LDH activity was increased in the supernatant of mEBs treated with ODQ at day 4, but then it returned to control value, indicating a relative toxicity caused by ODQ at the beginning of the treatment, which however disappeared shortly thereafter. All other treatments did not significantly influenced LDH activity.

  1. Supplementary Material