Knockdown of GRP78 sensitizes human leukemia cells to AraC. (A) Western blot analysis of NB4 cells transfected with pcDNA or Flag-tagged GRP78 expression vector and then treated with the indicated AraC concentration for 24 hours for detection of cleaved caspase-7 and PARP with β-actin as loading control. (B) Quantitation of cleaved caspase-7 and PARP normalized to β-actin in panel A. The ratio at 0μM AraC in pcDNA cells was set as 1. (C) Western blot analysis of lysates from NB4 cells transfected with siGrp78 or sictrl and treated with the indicated AraC concentration for 24 hours for detection of cleaved caspase-7 and cleaved PARP with β-actin as loading control. (D) Quantitation of cleaved caspase-7 and PARP normalized against β-actin in panel C. The ratio at 0μM AraC in sictrl cells was set as 1. (E) Representative annexin V/7-AAD flow cytometric apoptosis analysis of HL60 cells transfected with control siRNA (sictrl) or siRNA against Grp78 (siGrp78). The cells were either nontreated (0 hours) or treated with 10μM AraC for 24 hours. (F) Time course analysis of apoptotic HL60 cells treated with AraC. HL60 cells were transfected with sictrl or siGrp78, followed by treatment with 10μM AraC for the indicated time (hours). The percentage of apoptotic cells was measured by annexin V/7-AAD flow cytometry. The level of apoptosis at 0 hours in cells treated with sictrl was set as 1. Data are mean ± SE. *P < .05 (Student t test).