Consistent with the phenotype, cells stably expressing both G9a and G9a4KR exhibited reduced levels of myogenic differentiation markers Myogenin and Troponin T (Fig. 2d).
These include efficiency of re-plating from single cells, growth rate, dependence on exogenous growth factors, reduced levels of spontaneous differentiation, colony appearance, apoptosis and in some cases CD30 surface marker expression [1], [4], [5], [6], [8], [9], [10], [11].
Mouse C2C12 myotubes treated with limb regeneration extracts were able to induce myotubes to reenter the cell cycle, exhibited reduced levels of muscle differentiation proteins and cleaved to produce smaller myotubes or proliferating mononucleated cells [ 15].
Reduced levels of induction of differentiation markers was also observed in organotypic raft cultures, where knockdown of p300 resulted in reduced expression of K1 (early marker) and filaggrin (late marker) (Figure 2A).
Especially the large 1.5 MDa supercomplex displays reduced levels of complex IV after differentiation.
DOI: http://dx.doi.org/10.7554/eLife.01557.027 10.7554/eLiFigure57.028 Figure 6 figure supplement 2. Reduced levels of p400 binding to differentiation genes upon Hdac6 KD.
DOI: http://dx.doi.org/10.7554/eLife.01557.026 10.7554/eLiFigure57.027 Figure 6 figure supplement 1. Reduced levels of Tip60 binding to differentiation genes upon Hdac6 KD. (Left) Example genomic loci showing Tip60 binding as measured by ChIP-seq upon GFP KD or Hdac6 KD.
To validate our transgenic model, we also assayed flx satellite cells, which, as expected, showed reduced levels of Pax7 and increased spontaneous differentiation after tamoxifen addition (Supplementary Figures 14a and b).
Cells transfected with Oct4 RNAi constructs demonstrated reduced levels of Oct4 mRNA and exhibited characteristics of trophectodermal differentiation.
