The cytokinesis block proliferation index (CBPI) assay

Myeloma cell lines were analyzed for regulation of mitotic entry by CBPI assay as described previously [34, 35]. Briefly, cells treated or not with E64d, were exposed to UV-C (2,5 jm⁻²) and subsequently incubated for 72 h with Cyt-B. After cytospin, the cell preparations were stained with Giemsa solution. The cells can be analyzed for their mitotic status (mononucleated, binucleated, multinucleated). Every analyzed condition was scored for the percentage of binucleated/mononucleated cells. Furthermore, to determine whether HO-1 could also influence the cellular response to DNA damage, following the CBPI assay, MM cells were treated for 24 h with E64d, exposed to UV and then incubated with CytB for 72 h. CytB allows the accumulation of dividing cells at the binucleated stage by inhibiting the rate of actin polymerization and the interaction of actin filaments. Therefore, these cells progressed through the G2/M checkpoint and mitosis but fail to divide due to presence of CytB [35]. Under physiological conditions, DNA damage leads cells activate the G2/M cell cycle checkpoint thus increasing the percentage of mononucleated cells in respect of the binucleated.