TDZ analogues act on t(6;11)-AML cells with reduced neuroleptic side effects. (A) Cell death (annexin V⁺, PI⁺, and annexin V⁺/PI⁺) evaluated 24 hours after treatment with TDZ and the analogues TDZ1, TDZ2, TDZ3, TDZ4, TDZ5, and TDZ6, compared with the DMSO value (n = 3). (B) Confocal immunofluorescence images of SHI-1 cells stained with F-actin antibody and with diamidino-2-phenylindole (DAPI) as the nuclear counterstain, seeded onto fibronectin (FN)-coated slides 4 hours after treatment with TDZ or its analogues (n = 2). Original magnification ×63. Bar represents 10 μm. (C) Live intracellular Ca²⁺ measurement in cells loaded with Fluo-4 AM, a Ca²⁺ indicator, measured by flow cytometry in Ca²⁺-containing or in Ca²⁺-free buffer. TDZ and TDZ analogues (or DMSO as the control) were added after the basal measurement at time 0 (n = 2), and fluorescence was detected 1, 3, and 5 minutes after treatment with TDZ. (D) Stepped-dose test (evaluated as the number of steps per second) performed 1 hour after TDZ, TDZ2, or TDZ6 treatment in NSG mice at the quantities shown (n = 3). (E) Tumor area in mice receiving a flank injection of SHI-1 cells and treated daily with TDZ 8 mg/kg or TDZ-equivalent molar doses of TDZ and TDZ6, compared with the control group treated with DMSO (n = 5), at day 31 after injection, after 20 days of treatment. Gray area indicates treatment interval. Data are the mean ± SEM. *P < .05; **P < .01; ***P < .001; ****P < .0001.