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. 2016 May 26;44(17):8129–8143. doi: 10.1093/nar/gkw483

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© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 7. — Modulation of cell cycle genes blocks proliferation and rescues myogenic differentiation in G9a overexpressing cells. (A) pBabe-G9a cells were transfected with control or CyclinD1 siRNA (siCyclinD1) in growth medium for 48 hr. The knockdown of CyclinD1 in pBabe-G9a cells was examined by western blot. β-actin was used as an internal control. (B) Control and siCyclinD1 pBabe-G9a cells were pulsed with BrdU and analysed by immunofluorescence with anti-BrdU antibody. The percentage of BrdU⁺ cells was quantified. Results are representative of two independent experiments. Error bars indicate mean BrdU⁺ cells obtained by counting at least 500 cells in each experiment ± SD. The reduction in S-phase population was also verified by PI staining of control and siCyclinD1 cells. (C) pBabe-G9a cells were transfected with Flag-p21 and Flag-Rb1 individually and their expression was analysed by western blot using anti-Flag antibody. (D) pBabe and pBabe-G9a cells transfected with p21 and Rb1 were differentiated for 24 hr and lysates were analysed for myogenin and Troponin-T by western blot. (E) Cells were immunostained with anti-MHC antibody (red) 36 hr after differentiation. Myogenic index was quantified and is shown in the right panel. The results are representative of two independent experiments. Error bars indicate the mean myogenic index ± SD.