Galactose Effects on Human Muscle Cell Metabolism Figure 3. Effect of replacing a glucose medium with a galactose medium on mitochondrial markers. A. Mitochondrial yield measured in myotubes differentiated for 7 days in HG (25 mM glucose), LG (5 mM glucose) or GAL (10 mM galactose). Mitochondrial yield was determined as mitochondrial protein content per total cellular protein content. Results are presented as means 6 SEM, n = 6, in which each condition was assessed in duplicate. B. Cardiolipin staining in myotubes differentiated for 7 days in HG (25 mM glucose), LG (5 mM glucose) or GAL (10 mM galactose). Cardiolipin was stained with 10N-nonyl acridine orange (NAO, 1 mM) in fixed myotubes for 30 min. The intensity of the staining was then measured by fluorescence. Results are presented as means 6 SEM, n = 3. C. Citrate synthase activity measured on isolated mitochondria from myotubes differentiated for 7 days in HG (25 mM glucose), LG (5 mM glucose) or GAL (10 mM galactose). Results are presented as means 6 SEM, n = 5, in which each condition was assessed in duplicate. D. Left panel: representative Western blot of Complex III and SDHA expression in myotubes differentiated for 7 days in HG (25 mM glucose), LG (5 mM glucose) or GAL (10 mM galactose). Complex III and SDHA were used as a mitochondrial content marker and beta-actin as a loading control. Right panels: quantification by densitometry of complex III and SDHA expressions. Data are presented normalized to beta-actin expression. Data are shown as mean 6SEM, n = 4. E. Cytochrome C oxidase activity measured in isolated mitochondria from myotubes differentiated for 7 days in HG (25 mM glucose), LG (5 mM glucose) or GAL (10 mM galactose). Results are presented as means 6 SEM, n = 5, in which each condition was assessed in duplicate. *, p,0.05, GAL vs HG and LG. F. Top panel: representative Western blot of Complex IV expression in myotubes differentiated for 7 days in HG (25 mM glucose), LG (5 mM glucose) or GAL (10 mM galactose). Beta-actin was used as a loading control. Bottom panel: quantification by densitometry of complex IV expression. Data are presented normalized to beta-actin expression. Data are shown as mean 6SEM, n = 4. *, p,0.05, GAL vs HG and LG. G. Left panel: representative Western blot of P-AMPK expression in myotubes differentiated for 7 days in HG (25 mM glucose), LG (5 mM glucose) or GAL (10 mM galactose). Beta-actin and AMPKa2 were used as loading controls. Right panels: quantification by densitometry of P-AMPK. Data are presented normalized to beta-actin and AMPKa2 expression. Data are shown as mean 6SEM, n = 3. *, p,0.05, GAL vs HG and LG. doi:10.1371/journal.pone.0028536.g003 PLoS ONE | www.plosone.org 5 December 2011 | Volume 6 | Issue 12 | e28536