FIGURE 3.

PLD activity is required for in vitro myogenic differentiation. A, [³H]palmitic acid-labeled cells were pretreated for 15 min by 1% 1-butanol and various PLD inhibitors: non isoform-specific fluoro-2-indolyldeschlorohalopemide (FIPI), PLD1-specific I-PLD1, and PLD2-specific I-PLD2. Tritiated phosphatidylbutanol (Pbut) formed was quantified after 10 min of stimulation by 10⁻⁷ m AVP. PLD activity is expressed as the percentage of radioactivity in phosphatidylbutanol relative to radioactivity in total phospholipids (*, different from AVP-stimulated cells, p < 0.05). B, L6 cells were cultured in the presence of 10⁻⁷ m AVP for 48 h with varying concentrations of PLD inhibitor fluoro-2-indolyldeschlorohalopemide. Cells were lysed, and proteins were subjected to Western blot analysis of myogenin expression. C, L6 cells were cultured in the presence of 10⁻⁷ m AVP for 48 h, with varying concentrations of PLD1-specific inhibitor (left panel) or PLD2-specific inhibitor (right panel). Cells were lysed, and proteins were subjected to Western blot analysis of myogenin expression (mean ± S.E. of three determinations; *, significantly different from cells + AVP, p < 0.05). D, shown is immunofluorescence microscopy of nuclear myogenin in non-infected L6 cells (NI) and cells infected with adenovirus coding for GFP, hPLD1 (PLD1), or hPLD2 (PLD2) and cultured in the presence of AVP for 48 h. Total nuclei were visualized by DAPI staining. Differentiation was assessed by the mean percentage of myogenin-positive nuclei, determined in 10 fields. *, significantly different from control cells, p < 0.001. Bar = 40 μm.