Fig. 1. Calcineurin, CaMK, p38-MAPK and PI3K regulatory pathways are involved in human myoblast differentiation. (A) Induction of myogenic differentiation requires a membrane hyperpolarization. Neither myogenin nor MEF2 were detected in myoblast maintained in growing medium (GM). Myoblasts were induced to differentiate for 3 days either in differentiation medium (DM) or in DM in which 116 mM Na⁺ was replaced by equivalent concentration of K⁺ to prevent the hyperpolarization (KCl). After 3 days in high K⁺ differentiation medium, myoblasts were replaced for 3 more days in control differentiation medium (wash KCl) in which they differentiated nicely. Myogenin was revealed with Alexa488, MEF2 with Alexa546, and nuclei with DAPI. Scale bar: 20 µm. (B) Differentiation is expressed as a percentage of myogenin- and MEF2-positive nuclei. Myoblast were induced to differentiate for 3-4 days in differentiation medium (DM). Myoblast differentiation is partially inhibited by 10 mM Cs⁺ (Cs) to block the hyperpolarization, by 10 µM SB202190 (SB) to block p38-MAPK, by 30 µM KN93 (KN) to block CaMK, by 50 µM LY294002 (LY) to block PI3K, and by a combination of 7 µM cyclosporin A (CsA) and 5 µM FK506 (FK) to block calcineurin. Results are expressed as mean±s.e.m.