Figure 2

EPA reverses TNF-α-mediated interference with myoblast fusion. C2C12 cells were induced to differentiate in the presence or absence of TNF-α (20 ng/ml) and EPA (50 μM). EPA was added together with TNF-α as a co-treatment (EPA+TNF) or, alternatively EPA was administered alone for a 2 hour pre-treatment after which it was withdrawn and replaced by TNF-α alone in DM (pEPA+TNF). Incubations were continued for 48 hours or 5 days in DM with replenishment of EPA and TNF-α at media changes. A myogenic index (MI) of fusion was calculated from ten images of randomly chosen microscope fields for DAPI and MyHC stained cells from each treatment. The total number of nuclei and the number of nuclei incorporated into myotubes were counted (A). Myotube size heterogeneity was evaluated after 5 days from the same images by calculating the number of nuclei per myotube and classifying them arbitrarily to categories of small (2–5 nuclei), medium (6–9 nuclei) or large (>10 nuclei) myotubes (B). Data are expressed as means ± standard error of mean (SEM) from 3 independent experiments (*p < 0.05 v. respective control; ^#p < 0.05 v. TNF-α; **NS v. control).