Mechanism of action of SGN-CD33A against MDR⁺ HEL 92.1.7 AML cells. Cells were incubated with SGN-CD33A, SGD-1882, or a control compound (10 μM CIS, cisplatin) before processing for Western blot analyses of (A) γH2AX and (B) cleaved PARP, phospho-p53, phospho-Chk2, phospho-Chk1, and Bmf. To confirm equivalent protein loading, blots were assessed for levels of total H2AX, p53, and β-actin. Cells treated with SGN-CD33A, SGD-1882, nonbinding control ADC, and cisplatin were also evaluated for (C) caspase-3 activity, (D) annexin V staining, (E) mitochondrial membrane integrity (ΔΨm) by Mitocapture assay, and (F) cytochrome c release. Cells were processed 44 to 48 hours after initial exposure for Western blot analyses and caspase-3 activity and after 70 hours for cytochrome c release. In some experiments, cells were preincubated with a caspase inhibitor (Z-VAD-FMK, 0.5 μM) for 30 minutes before treatment with SGD-1882 or SGN-CD33A or nonbinding control. For annexin V and assay for mitochondrial integrity, cells were treated with 100 ng/mL SGN-CD33A or nonbinding control ADC, 1 nM SGD-1882, or 3 μM cisplatin. Data shown in Figure 3C-F represent mean ± SD of values from 2 to 3 experiments. One hundred nanograms per milliliter SGN-CD33A is the ADC equivalent for 1 nM SGD-1882.