H-2Kb-tsA58 mdx myoblasts (H2K mdx cells) were cultured and differentiated as described previously (22 (link)–32 (link, link, link, link, link, no link found, no link found, no link found, link, no link found)). For internalization experiment, fluorescein-(FAM)-labeled thiomorpholino ASO (TMO1-FL and PMO-FL, Table 1) was transfected at 100 nM when complexed with lipofectin transfection reagent (Thermo Fisher Scientific) at the ratio of 2:1 (w:w) (lipofectin:ASO) and used in a final transfection volume of 500 μL/well in a 24-well plate as per the manufacturer's instructions, except that the solution was not removed after 3 h. In addition, to assess the ability of TMO to internalize into cells without transfection reagent, TMO1-FL and PMO-FL were incubated with cells at 200 nM for 1, 3, and 5 d. After indicated time points, cell nuclei were stained with Hoechst (Sigma Aldrich) for 10 min and washed five times with PBS containing 10% fetal bovine serum before the images were captured using Olympus TS-100 inverted fluorescence microscopy system with the NIS-Elements software (Nikon Instruments Inc., Hilton, South Australia, Australia). Quantitation of average fluorescence intensity was performed using Image J software.