Fig. 2. Kir2.1 channel activation and myoblast differentiation are linked to a tyrosine dephosphorylation. (A) Myoblasts were induced to differentiate for 6 hours in the absence or presence of 10 µM bpV(Phen). Kir2.1 current was recorded during a 300 ms step to -120 mV from a holding potential at -60 mV. The histogram represents the fraction of myoblasts with Kir2.1 current density of increasing amplitudes (from 0 pA/pF to -6 pA/pF). Leak currents were estimated as in Fig. 1A. Cell capacitances were 33±3 pF (n=47) in DM 6 hours and 20±2 pF (n=19) in DM supplemented with bpV(Phen). (Right) Kir2.1 current density in control conditions and when bpV(Phen) is added. This histogram is an alternative representation of the data on the left. (B) Myoblasts were induced to differentiate for 4 hours in the absence or presence of 10 µM genistein. Kir2.1 current was measured and results presented as in A. Cell capacitances were 24±3 pF (n=22) in DM 4 hours and 24±3 pF (n=31) in DM supplemented with genistein. (Right) Kir2.1 current density in control conditions and when genistein is added. This histogram is an alternative representation of the data on the left. (C) Fusion index of myoblasts kept for 24 hours in DM (control conditions), or in DM supplemented with 10 µM bpV(Phen) or with 10 µM genistein during the first 4 hours. Fusion index represents the fraction of nuclei within myotubes. As genistein is prepared in DMSO, we verified that the final DMSO concentration (0.1%) had no effect on the fusion index (n=6; P=0.59, data not shown). Myoblasts were fixed with ice-cold methanol, and stained with Hematoxylin-Eosin. Two different clones were evaluated. Nuclei were counted in three randomly chosen microscope fields for each condition. One microscope field usually contains between 400 and 500 nuclei. A representative picture in each condition is shown. Arrows indicate clusters of nuclei in myotubes. Scale bars: 50 µm. An anti-phosphotyrosine immunoblot on myoblast protein extract shows that 10 µM genistein and 10 µM bpV(Phen) affect overall tyrosine phosphorylation.