Figure 1. ZHTc6-MyoD cell study.

(a) Almost all of the ZHTc6-MyoD cells differentiated to form myotubes after the removal of Dox (−). However, a small fraction of the cells formed colonies. The colonies of the undifferentiated cells were cultured in maintenance medium, and subsequently differentiated into muscle lineage cells after the removal of Dox (−). (b) The DNA microarray analysis showed that MyoD expression in colony-forming cells at 13 days after differentiation was 90 times that in undifferentiated ZHTc6-MyoD cells. The levels of Pax7 and dystrophin expression were similar. PTH1R expression in the colony-forming cells at 13 days after differentiation was 40 times that in undifferentiated ZHTc6-MyoD cells. (c) PTH1R expression in the myotubes was 13 times that in undifferentiated ZHTc6-MyoD cells. (d) RT-PCR analysis also detected PTH1R expression in colony-forming cells at 13 days after differentiation (13d-ZHTc6-MyoD) and myotubes, but it was not expressed in the undifferentiated colonies. G3PDH expression was used as a positive control. Marker, DNA molecular weight Marker X (0.07–12.2 kb; Roche Mannheim, Germany). (e) The ZHTc6-MyoD cells that were cultured in differentiation medium containing PTH (PTH+) differentiated into spindle-shaped cells on day 3, and fused on day 4. The cells that were cultured in differentiation medium without PTH (PTH−) differentiated into spindle-shaped cells on approximately day 4, and fused on day 5. Scale bar represents 50 μm. (f) The locations of PTH1R mRNA sequences to which the PTH1R siRNA 5 to 8 were complementary are indicated in the diagram. (g) Almost all of the cells transfected with the cell death control siRNA died. Most cells transfected with the PTH1R siRNA 6, 7, or 8 died on day 2. However, a few cells survived, and differentiated to myotubes on day 10. The cells transfected with PTH1R siRNA 5 survived, and differentiated in a manner similar to that observed for the negative control.