FIGURE 7:

PLD1 inhibitor blocks mature myotube formation in L6 myoblasts, which can be partially rescued by addition of LPC. (A) Near-confluent L6 myoblasts were treated with either DM plus DMSO or DM plus PLD1 inhibitor (4 μM) for 4 consecutive days. Then the medium was replaced with DM with or without 50 μM LPC for another 15 h. Severe retardation of mature myotube formation was observed in PLD1 inhibitor-treated cells, whereas LPC treatment partially reversed the PLD1 inhibition. L6 cells were immunostained with myosin and myogenin antibodies, and the nuclei were counterstained with DAPI. (B) The fusion indexes of mature and nascent myotubes were calculated in the treated cells (n = 3, *p < 0.05, **p < 0.01). (C, D) Primary myoblasts isolated from WT and PLD1^−/− muscle were differentiated for 4 d, and PLD1^−/− culture was then treated with or without 50 μM LPC for another 15 h before immunofluorescence staining for myosin and myogenin expression. The fusion indexes of mature and nascent myotubes were calculated (n = 3, *p < 0.05, **p < 0.01). Again, addition of LPC could partially reverse the PLD1^−/− fusion defect.