Figure legends
Figure 1. C26-derived CM induces atrophy in C2C12 myotubes
Schematic representation of the processes involved in differentiation of C2C12 myoblast into myotubes and the subsequent treatment with CM isolated from C26 colon cancer cells. (B ) Representative images obtained from immunofluorescence (40×) highlighting the myotube diameter upon treatment with C26-derived CM. Scale bars represent 20 μm. (C ) Quantification of myotube diameter (n=3). (D ) Western blot (80µg) showing a decrease in the expression of MyHC in C2C12 treated with C26-derived CM. β-actin was used as a loading control to confirm equal loading of protein. (E ) Metabolic activity of untreated and C26-dericed CM treated C2C12 myotubes at 24, 48, and 72 h. Absorbance was measure at 490 and 630 nm (n = 3). All data represented as mean ± s.e.m.; p-values as determined by two-tailed unpaired students’ t-test.Figure 2. Protein profile of C2C12 upon treatment with C26-derived CM
Schematic diagram depicting the step involved in mass spectrometry-based proteomics. (B) Venn diagram showing the total number of proteins identified between untreated and C26-dervied CM treated C2C12. (C) Quantitative Venn diagram portraying differential abundance of proteins identified in untreated and C26-dervied CM treated C2C12 cell lysates. (D) Volcano plot displaying the scatter of proteins identified in untreated and C26-dervied CM treated C2C12 cells. Curved lines represent significance threshold (n = 3). (E ) Heatmap showing statistically significant proteins with fold change greater than ≥2 identified in untreated and C26-dervied CM treated C2C12 cells.
Figure 3. Enrichment analysis of C26-derived CM treated C2C12
Gene enrichment analysis highlighting the biological processes that are enriched in C26-dervied CM treated C2C12 myotubes. (B)Gene enrichment analysis highlighting the biological processes that are depleted in C26-dervied CM treated C2C12 myotubes. (C ) Western blot (60 μg) showing the abundance of BiP and calnexin in C2C12 upon C26 CM treatment. β-actin was used as a loading control to confirm equal loading of protein. (D ) Relative intensity of Bip and calnexin in C26-derived CM treated C2C12 compared to untreated C2C12.n = 3; all data represented as mean ± s.e.m.; p-values as determined by two-tailed unpaired students’ t-test.