Fig. 4. Lbx1 expression in differentiating myoblasts and myotubes. Satellite cells were induced to differentiate in vitro following the method described in Materials and Methods, and then immunostained for Lbx1, myogenin and Pax7, and also counter-stained with Hoechst dye 33342 to identify nuclei. (A) Primary satellite cells were induced to differentiate into flat spindle-shaped cells by cultivating them at high density. Only some of the actively proliferating flat spindle-shaped cells express myogenin. Such myogenin-expressing cells (arrowheads) also express Lbx1, but at rather low levels. (B,C) Primary satellite cells were induced to differentiate into myotubes by cultivating them in differentiation medium for 5 days. The developing myotubes express both myogenin and Lbx1 but do not express Pax7 (arrows). The level of Lbx1 expression is rather low. Note that Pax7-expressing mononucleated cells (arrowheads) do not express Lbx1. Bar, 50 µm.