TNF-α activates caspase-3 in KG1a cells cocultured with stroma. (A) Cocultures of KG1a and HS5 cells were exposed to TNF-α (100 ng/mL) for 18 hours. All cells were collected after a brief exposure to trypsin, and CD45⁺ KG1a cells were isolated by MACS with magnetically labeled anti-CD45 antibody. Protein lysates of CD45⁺ cells were separated on 4-12% Bis-Tris gels and immunoblotted with a murine monoclonal anti–caspase-3 antibody that detects both full-length (32-kDa) and cleaved/active (17-kDa) caspase-3. The membrane was then stripped and immunoblotted with a rabbit anti–β-actin antibody. Only KG1a cells cocultured with HS5 showed active (cleaved) caspase-3 after TNF-α exposure (lanes 10-12). The pixel intensity of cleaved caspase-3 (relative to β-actin) in those lanes was 0.12 compared with less than 0.01 in lanes 1 to 9. (B) After treatment with TNF-α (100 ng/mL), cocultures of KG1a and HS5 cells were exposed to the FITC-conjugated caspase-3 inhibitor DEVD-FMK and labeled with anti–CD45-APC antibody. FACS analysis quantified the proportion of CD45⁺ cells with activated caspase-3.