Cell Growth, Proliferation, and Apoptosis Assessment

Growth curves were performed as previously described [41 (link), 46 (link)]; the number of viable cells was assessed on a TC-20 (Bio-Rad, Hercules, CA, USA). Cell cycle progression/proliferation was assessed by flow cytometry after BrdU/7AAD staining using the BrdU Flow Kit (BD Pharmingen, Franklin Lakes, New Jersey, USA) according to the manufacturer’s instructions and as described previously [41 (link), 46 (link), 50 (link), 51 (link)]. BrdU incubation was performed at a concentration of 50 µM for 1 h, and an LSR II machine (BD Biosciences) was used for flow cytometry. Apoptosis was assessed by Annexin-V/7AAD assays (BD Biosciences), as described earlier [41 (link), 46 (link), 50 (link), 51 (link)], using a CytoFLEX LX flow cytometer (Beckman Coulter, Brea, CA, USA).