Figure 3.

C₂C₁₂ myotubes parameters under Walker Factor effects and modulation by leucine treatment. (a) In vitro MTT assay for assessing cell mitochondrial activity in C₂C₁₂ myotubes subjected to Walker Factor and leucine supplementation. (b) Representative ultrastructure images of mitochondria from C₂C₁₂ myotubes. Groups without tumour presented a greater number of mitochondria, when compared to the other tumour-bearing groups (W and WL). The leucine supplemented tumour-bearing group (WL) had more mitochondria than the tumour-bearing group fed a control diet (W). Magnification 10000×. Scale bar: 2µm, arrows indicate mitochondria. Bar graphics show the mitochondrial number and area for each group. (c) Basal O₂ mitochondrial consumption; maximal mitochondrial respiration and spare respiratory capacity of the C₂C₁₂ myotubes measured in Oroboros Oxygraph-2K and DatLab software package (OROBOROS, Innsbruck, Austria). Experimental design applied on myotubes cells were sequentially treated with Oligomycin A (1 µM), FCCP (0.5 µM), and Antimycin A (1 µM) as indicated by the arrows. For more details see Material and Methods section. Leucine-treated C₂C₁₂ myotubes were incubated with leucine (50 μM, Sigma) for 24 h. Tumour-treated C₂C₁₂ myotubes were incubated for 24 h with Walker Factor at final concentrations of 25 μg/mL. Leucine/Walker Factor-treated C₂C₁₂ myotubes were exposed to 50 μM leucine and supplemented with tumour Factor at final concentrations of 25 μg/mL. For more details see Material and Methods section. Legend: control (C); leucine supplementation medium (L); Walker Factor treatment (WF) and Walker Factor treatment and leucine supplementation medium (WFL). * p < 0.05 difference against C group; ** p < 0.05 difference against L group: # p < 0.05 difference against WF group.