*3.3. Cell Proliferation by MTS Assay*

An MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay (Promega, Fitchburg, WA, USA) was used to determine the anti-proliferative effect of PN on CRC cell lines and its toxic effect on C2C12 muscle cells. For each cell line tested, 2 × 10<sup>4</sup> cells per well were seeded onto 96-well plates and allowed to attach. Cells were treated with PN at 0.1, 0.5, 1, 2, or 4 μg/mL and incubated for 48 and 72 h. Cetuximab of 30 μg/mL was added as a control. MTS solution (20 μL) was then added and cells were incubated at 37 ◦C for 1 h. Absorbance were measured at 490 nm (Model 550, Bio-Rad, Hercules, CA, USA). The experiments were performed in triplicate. IC50 values (concentrations that inhibited cell growth by 50%) were compared.
