Figure 2

Blocking nTRIP6 function accelerates early and delays late differentiation. Control untransfected C2C12 cells (Co) or cells transfected with either the mCherry-tagged, nuclear targeted nTRIP6 blocking peptide (BP) or the control, scrambled version of the peptide (cBP) were subjected to a differentiation experiment. (a) Twenty-four hours after transfection cell nuclei were counterstained with DRAQ7 and imaged by confocal microscopy. Representative images are shown. (b,c) Cells were harvested at the indicated day and the relative levels of the Myog (b) and Tnni2 (c) mRNAs were determined by reverse transcription and real-time PCR. Results are plotted relative to the expression of the Rplp0 gene (mean ± SD of three independent experiments). Bonferroni corrected P values are for (b) α = 0.044, β = 0.027 and γ = 0.012 and for (c) α = 0.050, β = 0.053 and γ = 0.047. (d–f) C2C12 cells transfected with the BP or the cBP were harvested at the indicated day of a differentiation experiment. Cell lysates were subjected to Western blotting using the indicated antibodies. Representative blots are shown. Full-length blots are presented in Supplementary Fig. S5. The expression of MYOG, TNNI2 and MYH3 relative to the β-actin loading control are presented as mean ± SD of three independent experiments.