*3.9. Cell Cycle Analysis*

A cell cycle evaluation experiment was carried out as described previously [44]. Briefly, 106 cells were treated with 30 μg/mL of C3 or C3–5 for 24 h. At the end of treatment time, cells were trypsinized, harvested and centrifuged at 2000 rpm for 5 min at 4 °C. Cells were washed with cold PBS and fixed with 70% EtOH for 1 h at −20 °C. Cells were incubated with 0.1% NP-40 on ice for 5 min, followed by 30 min of incubation on ice with 100 μg/mL RNase (Sigma-Aldrich, St. Louis, MO, USA). Finally, 50 μg/mL propidium iodide (PI) was added to cells for 20 min. Cell cycle analysis was carried out by flow cytometry using a FACSCantoII with FACSDiva software (Becton Dickenson, San Jose, CA, USA); 10<sup>4</sup> cells were counted for each the control and the treatment groups.
