Activation of CAR promotes CPA-mediated apoptosis of HL-60 cells in the HPH-HL-60 coculture model. In the coculture system, cells were treated with vehicle control (0.1% DMSO) or CPA (250, 500, and 1000μM) in the presence and absence of CITCO (1μM) as outlined in “Methods.” CPA-mediated apoptosis in HL-60 cells was analyzed with different assays. (A) Representative visualization of HL-60 cells stained with Hoechst 33342. Arrowhead and arrow depict normal nuclei and apoptotic bodies, respectively. (B) Quantitation of apoptotic bodies induced by CPA with/without CITCO. Two hundred HL-60 cells from each treatment group were calculated under fluorescent microscopy. Percent of apoptotic cells were expressed as mean ± SD obtained from 3 independent experiments (**P < .01). (C) DNA extracted from treated cells was loaded on an agarose gel to illustrate CPA-induced DNA fragmentation. (D) Caspase 3 activity was analyzed with Western blotting to detect the large fragment (17/19 kDa) of activated caspase 3 in HL-60 cells. β-actin was used to normalize protein loading. (E) Effects of CAR activation on CPA-mediated membrane translocation of phosphatidylserine during apoptosis were analyzed using flow cytometry as detailed in “Methods.”