Ca2+overload is toxic for t(6;11)AML cells. (A) Live intracellular Ca2+ measurement in SHI-1 cells loaded with Fluo-4 AM Ca2+ indicator, measured by flow cytometry. EGTA pretreatment was performed 30 minutes before TDZ, which was added at time 0 after the basal measurement. Fluorescence was detected 1, 3, and 5 minutes after TDZ treatment (n = 3). (B) Overlapping representative histograms showing mitochondrial depolarization evaluated by tetramethylrhodamine ethyl (TMRE) fluorescence measurement 24 hours after TDZ, EGTA, or EGTA+TDZ treatment (n = 3). (C) Cell death (annexin V+, PI+, and annexin V+/PI+) evaluated 6 and 24 hours after TDZ, EGTA, or EGTA+TDZ treatment, compared with the DMSO value (n = 2). (D) Histograms showing the percentage of elongated cells (n = 46-146 cells) and the percentage of cells containing F-actin aggregates (n = 156-408 cells), 4 hours after TDZ, EGTA, or EGTA+TDZ treatment. (B-D) EGTA pretreatment was performed 30 minutes before TDZ was added. (E) Representative overlapping histograms showing mitochondrial depolarization evaluated by TMRE measurement 6 and 24 hours after treatment with TDZ, KB-R7943, or KB-R7943+TDZ (n = 5). (F) Cell death (annexin V+, PI+, and annexin V+/PI+) evaluated 6 and 24 hours after TDZ, KB-R7943 or KB-R7943+TDZ treatment, compared with the DMSO value (n = 2). Data are the mean ± SEM. *P < .05; **P < .01; ***P < .001; ****P < .0001. ns, not significant.