Fig. 3. Effect of palmitate and/or unsaturated fatty acids on mitochondrial morphology. L6 myotubes were incubated for 16 h in media containing D-glucose (5 mM) supplemented with or without palmitate (PA, 0.4 mM) or unsaturated fatty acids palmitoleate (PO, C16:1), Oleate (OL, C18:1) or linoleate (LO, C18:2) at 0.4 mM as indicated prior to: (A) mitotracker green (mitospy) staining and live cell imaging by confocal microscopy of L6 myotubes (the scale bar represents 5 μm); (B) analysis of mitochondrial morphology (elongated versus fragmented) was quantified using ImageJ software and presented as elongated/tubular if mitochondria were >1 μm or fragmented if <1 μm in length and (C) Immunoblotting and quantification of mitofusin 2 (MFN2), a key regulator of mitochondria dynamics in a mitochondrial-enriched fraction. The abundance of TOM20 a mitochondrial marker protein was used as a gel loading control. The bar graph represents quantitative analysis of MFN2 abundance relative to TOM20 with data presented as mean + SEM from a minimum of 4 separate experiments. The asterisk indicates a significant change (P<0.05) to the control condition (lacking any fatty acid additions).