Figure 1.

The AP-HNS cell-permeable peptide enhances muscle cell differentiation and increases the accumulation of HuR in the cytoplasm. (A) C2C12 cells grown to confluency (d0) or induced to differentiate for 4 d (d4) were fixed and used for immunofluorescence, using the monoclonal anti-HuR antibody. DAPI staining was used to visualize the nuclear compartment. DAPI and HuR images of a single representative field of view are shown for each time point. (B) The subcellular localization of HuR after treatment of exponentially growing C2C12 cells with AP-HNS, AP-scHNS (scrambled HNS) or no peptide was analyzed. 24 h after peptide treatment. Myoblast cells were fixed, and the localization of endogenous HuR was determined by double immunofluorescence staining using a monoclonal anti-HuR antibody and DAPI. DAPI, HuR, and a merged image of a single representative field of view are shown for each treatment. (C) Exponentially growing C2C12 cells were treated with AP-conjugated peptides for 24 h, and differentiation was induced by serum starvation. AP-conjugated peptides were added with differentiation medium for another 24 h. Four days after differentiation induction (day 4), cells were fixed and stained for myoglobin. The degree of differentiation after each peptide treatment was analyzed by immunofluorescence. Myoglobin-stained images of a single representative field of view for each peptide treatment are shown. (D) Immunofluorescence staining with the anti-myoglobin antibody was used to calculate the fusion index of the myotubes after each peptide treatment. The quantitative analysis of differentiation was performed by determining the number of nuclei in each microscopic field in relation to the number of nuclei in myotubes in the same field. The percentage of differentiation is indicated. Error bars, SD of two independent experiments.