Fig. 2

Generation and validation of biologically relevant myoblast line show effective reporting of sarcospan gene activity. The promoter region of the muscle-specific human sarcospan (hSSPN) gene was predicted using UCSC genome browser gene regulatory data. Using traditional cloning techniques, a A 2-kb region of the predicted hSSPN promoter was amplified and inserted into a promoterless EGFP plasmid. The construct was used to transfect C2C12 murine myoblasts reporter cell lines, which were then selected for stable transfection using antibiotic selection. Confluent myoblasts (day 0, D0) were switched from proliferation to differentiation media and assayed every other day for 8 days (D2 to D8). b, c High-content imaging of differentiating hSSPN-EGFP cells showed a transition to mature myotube morphology that correlates with an increase in reporter signal (n = 88 per time point). Fluorescence values are reported as relative fold change over myoblast values (R.U., relative units). Scale bar, 100 μm. ****p < 0.0001