*4.10. Cell Cycle Analysis*

Cell cycle distribution was evaluated 24 h after cell treatment with the respective IC<sup>50</sup> of GSH-NS B or GSH-NS D. The occurrence of the so-called sub-G0/G<sup>1</sup> peak, which is a distinct cell population characterized by subdiploid DNA fluorescence and might correlate with the internucleosomal DNA fragmentation typical of apoptosis (Pozarowski and Darzynkiewicz, 2004), was also evaluated. Briefly, 1 × 10<sup>6</sup> HCT116, 1 × 10<sup>6</sup> HT-29, 1 × 10<sup>6</sup> DU145, and 1 × 10<sup>6</sup> PC-3 cells were incubated with 2 µM of the live cell staining Vybrant Dye Cycle Green (Invitrogen) for 30 min at 37 ◦C. The samples were run on a flow cytometer with 488 nm excitation to measure Vybrant Dye Cycle Green staining and data analysis was performed by FCS Express software version 4 (BD Bioscience, Milano, Italy).
