Figure 5.

CRC cells are sensitive to arginine deiminase treatment. (A) Dose-Response Non-Linear Regression Curves and IC₅₀ values of the indicated CRC cell lines treated with the ADI-PEG20. The percentage (%) of cell growth was calculated relative to the cell numbers in corresponding PBS-treated control samples, which was selected as 100%. IC₅₀ values were obtained from non-linear regression analysis of concentration of the drug vs response curves. The results were obtained from three independent experiments. Quadruplicate samples were assessed for cell growth after a 6-day period of treatment by cell counting for each individual experiment. The error bars represent ±SEM. (B) Representative flow cytometry scatterplots of EdU incorporation in HCT116, RKO, HT29 and SW480 CRC cell lines after 72 h treatment with ADI-PEG20 (1 μg/mL) or PBS-vehicle control. EdU was measured using the using Click-iT® EdU Alexa Fluor® kit and total DNA stained using FxCycle™ Violet Stain. (C) Quantification of EdU incorporation from three independent experiments. Data are presented as mean ± SEM. *P < 0.05, ****P < 0.0001, two-tailed t-test. (D) Western blot analysis of the cell cycle markers Cyclin D1 and D3 and the urea cycle enzyme ASS1 in cells treated for the indicated time with ADI-PEG20 (1 μg/mL). Actin was used as endogenous loading control. Original western blots are reported in Supplementary Fig. S15. (E) Quantification of Cyclin D1 and D3 protein expression from triplicate experiments. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-way ANOVA.