LIF inhibits activation of caspase-3 and DNA fragmentation during differentiation. (A) Representative immunofluorescent images of 10 ng/mL LIF-treated and control cultures labelled for cleaved caspase-3 (green) and DNA with DAPI (blue). A cleaved caspase-3 positive myoblast with more typical apoptotic morphology with condensed chromatin and small cytoplasmic space is indicated with a thick arrow. A cleaved caspase-3 positive myoblast with normal fusiform morphology is indicated with a thin arrow. Scale bar is 100 μm. (B) Cultures differentiated for 24 hours and immunofluorescently stained for cleaved caspase-3 and MYHC simultaneously were counted and the percentages of cleaved caspase-3 positive nuclei (of total nuclei) and fusion index (percentage of nuclei contained within MYHC positive syncytia) represented graphically. -/+ indicates either the absence or presence of LIF (10 ng/mL), wortmannin (WORT; 100 nM), U0126 (10 μM) or DMSO (0.1% (v/v) in media as the vehicle for wortmannin and U0126) treatment. Asterisk (*) indicates P < 0.05 and ns indicates P > 0.05 with one-way ANOVA and Dunnet's post-hoc test for all treatments compared to control (n = 4). (C) After 24 hours of differentiation cultures were TUNEL stained and the percentage of total cells positive for TUNEL represented graphically. Asterisk (*) indicates P < 0.05 for LIF-treated compared to control with Student's t-test (n = 4).