HAP1 loss promotes leukemic cell survival and prevents apoptosis through downregulation of the Ca²⁺-mediated calpain-1-Bid-caspase-3/12 apoptotic pathway. (A) SEM control cells or depleted of HAP1 were treated with l-asparaginase (10 mIU/mL) for 24 hours. Cell lysates were subjected to SDS-PAGE and immunoblotted (left) for HAP1, and intact and cleaved calpain-1, Bid, caspase-3, caspase-12 and PARP1, and actin. Representative blots from 3 independent experiments showing similar result patterns are shown. Charts on the right show the densitometry analysis (National Institutes of Health Image J 1.61) of blots from 3 independent experiments. Apoptotic and necrotic populations of cells double-stained with PI- and FITC-labeled Annexin V were assessed by flow cytometry. (B) SEM control cells are more vulnerable to apoptosis on l-asparaginase treatment compared with (C) HAP1-depleted cells. Apoptosis and necrosis were assessed after 12 hours of 10 mIU/mL of l-asparaginase treatment as described in “Methods and materials.” Values are means ± standard error of the mean from 3 independent experiments. **P < .05.