Fig. 3.

Propagation of large RFP-cNLS fusion proteins is restricted by the classical nuclear import pathway. Myoblasts were transfected with inducible vectors for RFP-cNLS fusion proteins and differentiated for 2 d. (A) After differentiation, doxycycline was added to the culture medium with or without 5 µM importazole, an inhibitor of importin-β, and the distributions of RFP within individual Hoechst-stained myotubes were detected by epifluorescence microscopy. (B–D) The effect of importazole treatment on myonuclear propagation was quantified in myotubes expressing mCherry-cNLS (B), tdTomato-cNLS (C), and DsRed-cNLS (D). Distances and fluorescence intensities were normalized to the brightest myonucleus within each myotube. Data are binned by distance (bin size, 50 µm) and are represented as mean ± SEM (two-way ANOVA with Bonferroni posttest). The dotted line represents the approximate boundaries of the transfected myotubes. (Scale bars: 100 µm.) *P < 0.05; **P < 0.01; ***P < 0.001.