Fig. 8. Expression of muscle-specific markers in ryanodine-treated primary cultures. (A) Western blot analysis of protein extracts prepared from embryonic, fetal and satellite muscle cells, cultured for 72 hours in the absence of ryanodine (C) or treated with ryanodine (Ry) for the indicated time periods in culture. At the end of 0-24 hours and of 24-48 hours of treatment, ryanodine was removed and the cells cultured for an additional 48 and 24 hours, respectively. The blots were reacted with the anti-MyHC mAb MF20 and with the anti-myogenin mAb F5D. An anti-tubulin antibody was used to normalize the blot. (B) RT-PCR analysis of total RNA isolated from fetal muscle cells cultured for 72 hours in the absence of ryanodine (C) or treated with ryanodine in the 24-48 hours time period in culture, after which ryanodine was removed and the cells cultured for the additional 24 hours. 100 ng of total RNA were reverse-transcribed and PCR-amplified using primers specific for MyHC, MyoD, myogenin and myf5, as indicated; by increasing the PCR cycles (over 30 cycles), an amplified product for MyHC becomes detectable also in ryanodine-treated cells (data not shown); primers specific for ß-actin were used to normalize the reaction. The experiments shown here were performed using 100 µM ryanodine; same results were obtained using 300 µM ryanodine (data not shown).