Zalypsis stimulates a p53-dependent DNA damage response through the induction of DSBs. (A) MM1S and OPM1 cells were treated with 5 nM Zalypsis for the indicated time points, cell protein extracts were obtained of each condition, and the phosphorylation of H2AX and CHK2 was analyzed by Western blotting. Equal loading was confirmed with an anti-GAPDH antibody. (B) Western blot showing the basal protein levels of p53 protein in the 9 MM cell lines tested. The 3 first cell lines are very sensitive to Zalypsis, whereas the 6 last ones are more resistant. Equal loading was confirmed with an anti-GAPDH antibody. (C) All the 9 MM cell lines were treated with 5 nM Zalypsis for 0, 3, 6, and 12 hours, and the induction of p53 protein levels was analyzed by Western blotting. Equal loading was confirmed with an anti-GAPDH antibody. (D) MM1S cells were treated with different antimyeloma agents at different doses (dexamethasone 1 μM, melphalan 10 μM, doxorubicin 100 nM, bortezomib 10 nM, and lenalidomide 10 μM) for 0, 3, 6, 12, and 24 hours, and changes in p53 protein levels compared with treatment with 5 nM Zalypsis for 12 hours were analyzed by Western blotting. (E) Western blot showing changes in the protein levels of some p53 targets (Bax and Noxa) after treatment of MM1S cells with 5 nM Zalypsis for different time points.