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Tissue-Specific Stem Cells
Version of Record online: 27 NOV 2012
Copyright © 2012 AlphaMed Press
Volume 30, Issue 12, pages 2619–2630, December 2012
How to Cite
Marchildon, F., Lala, N., Li, G., St-Louis, C., Lamothe, D., Keller, C. and Wiper-Bergeron, N. (2012), CCAAT/Enhancer Binding Protein Beta is Expressed in Satellite Cells and Controls Myogenesis. STEM CELLS, 30: 2619–2630. doi: 10.1002/stem.1248
F.M.: conception and design, data analysis and interpretation, manuscript writing, and final approval of manuscript; N.L., G.L., and D.L.: collection and assembly of data, data analysis and interpretation, and final approval of manuscript; C.S.: administrative support, collection and assembly of data, and final approval of manuscript; C.K.: provision of study materials, manuscript writing, and final approval of manuscript; N.W.: conception and design, financial support, data analysis and interpretation, manuscript writing, and final approval of manuscript.
Disclosure of potential conflicts of interest is found at the end of this article.
First published online in STEM CELLSEXPRESS November 7, 2012.
- Issue online: 27 NOV 2012
- Version of Record online: 27 NOV 2012
- Accepted manuscript online: 3 OCT 2012 03:16PM EST
- Manuscript Accepted: 4 AUG 2012
- Manuscript Received: 7 FEB 2012
- Canadian Institutes of Health Research (CIHR)
- Cancer Research Society with funds from the University of Ottawa's Research Development Program
- Ontario Graduate Scholarship
Additional Supporting Information may be found in the online version of this article.
|sc-12-0136_sm_SupplFigure1.tif||956K||Figure S1. Overexpression of C/EBPβ in myoblasts does not affect cell growth. (A) Crystal violet assay to determine cell number of satellite cell cultures retrovirally transduced to express C/EBPβ or with empty vector (pLX) and cultured for 48 hours in growth medium. Error bars are the SEM, n=3. (B) Crystal violet assay to determine cell number of C2C12 cells retrovirally transduced to express C/EBPβ or with empty vector (pLX) and cultured for 48 hours in growth medium. Error bars are the SEM, n=4.|
|sc-12-0136_sm_SupplFigure2.pdf||96K||Figure S2. Restoration of MyoD protein expression rescues the differentiation defect in C/EBPβ-overexpressing C2C12 cells. (A) Indirect immunostaining for MHC expression in C2C12 cells retrovirally transduced to express C/EBPβ or with empty vector (pLXSN), and transiently transfected to express GFP and MyoD as indicated. Cells were differentiated in low serum conditions for 4 days prior to immunostaining. DAPI staining reveals nuclei. Scale bar = 50μm. (B) Differentiation index of cultures treated as in (A). *p<0.05, n=3. Error bars are the standard deviation. (C) Western analysis of C/EBPβ, MyoD and myogenin expression in C2C12 cultures transfected and differentiated as in (A) β-tubulin is used as a loading control. (D) Images of GFP expression, merged with phase contrast images, to determine transfection efficiency. (E) Transfection efficiencies, defined as the percentage of GFP+ cells in the culture 24 hours after transfection, for each transfection condition. One way ANOVA analysis revealed a significant decrease (p<0.05) between conditions labeled a and b.|
|sc-12-0136_sm_SupplFigure3.tif||1080K||Figure S3. Loss of MyoD expression is partially rescued by the inhibition of the proteasome. (A) Western analysis of C2C12 myoblasts retrovirally transduced to express C/EBPβ or with empty vector (pLXSN) and cultured in 2% horse serum for 36 hours. Two hours prior to harvest, cells were treated with 50 μM MG132. Cyclophilin B is used as a loading control. (B) Western analysis of C2C12 myoblasts retrovirally transduced to express C/EBPβ or with empty vector (pLXSN) and cultured in growth medium pretreated with MG132 for 2 hours prior to harvest.|
|sc-12-0136_sm_SupplFigure4.tif||535K||Figure S4. Withdrawal of HGF from growth medium reduces Cebpb expression and promotes differentiation. (A) RT-qPCR analysis of Cebpb, Pax7 and Myog expression in WT satellite cell cultures grown in complete growth medium or growth medium lacking HGF for 48 hours. *p<0.05, **p<0.01, n=4. (B) Western analysis of C/EBPβ and Pax7 expression in SCs cultured and treated as in (A).|
|sc-12-0136_sm_SupplFigure5.pdf||113K||Figure S5. Fiber hypertrophy increased at day P56 in conditional knockout mice. (A) Representative bright field images of tibialis anterior cross sections from control Cebpbfl/fl and conditional null Cebpb-/-Pax7Cre/+ animals at postnatal day 56 stained with hematoxylin and eosin. Scale bar = 100 μm. (B) Average cross-sectional areas of muscle fibers from control and conditional null animals. For each group n=2 animals (1 male and 1 female). Error bars are the standard deviation. (C) Relative fiber number in the tibialis anterior muscles of control and conditional null mice (n=2 for each group). Control animals are set arbitrarily at 100. (D) Frequency distribution of fiber size in control and conditional null mice. Error bars are the standard deviation.|
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