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Embryonic Stem Cells/Induced Pluripotent Stem Cells
Version of Record online: 19 DEC 2012
Copyright © 2012 AlphaMed Press
Volume 31, Issue 1, pages 59–70, January 2013
How to Cite
Magli, A., Schnettler, E., Rinaldi, F., Bremer, P. and Perlingeiro, R. C. R. (2013), Functional Dissection of Pax3 in Paraxial Mesoderm Development and Myogenesis. STEM CELLS, 31: 59–70. doi: 10.1002/stem.1254
Author contributions: A.M.: designed and performed research, analyzed data, and wrote the manuscript; E.S., F.R., and P.B.: performed research and analyzed data; R.C.R.P.: contributed with interpretation of the data and wrote the paper.
Disclosure of potential conflicts of interest is found at the end of this article.
First published online in STEM CELLSEXPRESS October 18, 2012.
- Issue online: 19 DEC 2012
- Version of Record online: 19 DEC 2012
- Accepted manuscript online: 18 OCT 2012 07:33AM EST
- Manuscript Accepted: 8 SEP 2012
- Manuscript Received: 9 JUL 2012
- NIH. Grant Numbers: R01 AR055299, RC1AR058118
Additional Supporting Information may be found in the online version of this article.
|sc-12-0552_sm_SupplFigure1.tif||943K||Figure S1. A) Gene expression levels of Pax3, Pax7 and Myf5 in inducible Ires-GFP day 5 control EBs. qRT-PCR was performed using the indicated TaqMan probes and the results were normalized to Gapdh endogenous levels. B) Paraxial mesoderm and myogenic marker are up-regulated by Pax3 induction in day 5 EBs. qRT-PCR was performed using the indicated TaqMan probes and the results were normalized to Gapdh endogenous levels. The graphs report the mean +SD for at least 3 independent experiments. C) Frequency of PDGFRα+FLK-1+ (P+F+), PDGFRα+FLK-1- (P+), PDGFRα-FLK-1+ (F+) and PDGFRα-FLK-1- (NEG) fractions before and after fluorescence activated cell sorting (FACS). Data are mean ±SD of 3 independent experiments.|
|sc-12-0552_sm_SupplFigure2.tif||2368K||Figure S2. Effect of Pax3 on MyoD and Myogenin expression levels at day 15 of differentiation. A) Schematic diagram showing the timing of Pax3 induction. At day 5, PDGFRα+FLK-1- cells were sorted and cultured until day 15. B-C) Western blot (B) and gene expression (C) analyses (n=2) for MyoD, MyoG and Pax3.|
|sc-12-0552_sm_SupplFigure3.tif||1426K||Figure S3. Alignment of the C-terminal region of PAX3 and PAX7 sequences from different organisms, adapted from the Conserved Domain Database (NCBI). The region in red represents the pfam12360.|
|sc-12-0552_sm_SupplFigure4.tif||617K||Figure S4. Gene expression analysis of Pax3 cell lines. RNAs were extracted from total EBs (day 5) using Trizol and used for cDNA synthesis. qRT-PCR was performed using the indicated TaqMan probes and the results were normalized to Gapdh endogenous levels and reported as relative expression to the non-induced sample. The graphs report the mean +SD for at least 3 independent experiments.|
|sc-12-0552_sm_SupplFigure5.pdf||115K||Figure S5. Pax3 represses cardiac terminal differentiation. Unsorted day 5 EBs noninduced or dox-induced from day 2 were trypsinized and plated as monolayer and cultured until day 10. A) Cells were fixed and analyzed by immunofluorescence staining with an anti-cTNI antibody, or B) harvested, lysed with RIPA buffer and analyzed with western blot using anti-MYOD, anti-cTNI and anti-GAPDH antibodies. Pax3 (lanes 1 and 6); Pax3.8 (lanes 2 and 7); Pax3 .PD-C (lanes 3 and 8); Pax3 .HD-C (lanes 4 and 9); Pax3 .PD (lanes 5 and 10). Bar: 50μm.|
|sc-12-0552_sm_SupplTable1.pdf||27K||Supplementary Table 1.|
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