Caspase activities and mitochondrial functions in DcR3.Fc-treated DCs. (A) Top panel shows effect of caspase inhibitor on DcR3-mediated apoptosis. CD14⁺ monocytes (5 × 10⁵) were pretreated with caspase inhibitors (general caspase, 50 μM Z-VAD-FMK; caspase-2, 50 μM Z-VDVAD-FMK; caspase-3, 50 μM Z-DEVD-FMK; caspase-8, 50 μM Z-IETD-FMK; and caspase-10, 50 μM Z-AEVD-FMK) for 1 hour before incubation with GM-CSF (200 ng/mL) and IL-4 (10 ng/mL) in the presence of 10 μg/mL Fc-fusion proteins or IgG₁. Cell viability was determined by incubation with annexin V–PE/7-AAD and analyzed by flow cytometry. *P < .05 compared with the control group. Bottom panels show determination of caspase activities using fluorescent substrates. Caspase activities in cell lysates were determined by incubation with fluorescent probes MCA-IETD.APK (DNP), MCA-AEVD.APK (DNP), MCA-VDVAD.APK (DNP), and MCA-DEVD.APK (DNP) to measure the activities of caspase-8 (top left), caspase-10 (top right), caspase-2 (bottom left) and caspase-3 (bottom right), respectively. *P < .05 compared with nontreated group. (B) Activation of caspases in DcR3.Fc-treated DCs. Cells were incubated with DcR3.Fc (10 μg/mL) for different time intervals before harvesting for Western blot analysis. Blots were probed with respective mAb to detect activated caspase-3 or caspase-8 and cleaved Bid or PARP. Vertical lines have been inserted to indicate a repositioned gel lane. (C) Determination of mitochondria membrane potential by incubation of DcR3-treated DCs with JC-1 (10 μg/mL) for 30 minutes before flow cytometric analysis (FL2 MFI). (D) Analysis of apoptotic cells by MTT at different time intervals. A total of 5 × 10⁴ monocytes were incubated with GM-CSF (200 ng/mL) and IL-4 (10 ng/mL) in the presence of (10 μg/mL) Fc-fusion proteins or IgG₁, followed by addition of MTT for 4 hours and SDS for another 16 hours. MTT formazan was measured as described in “Methods.” Error bars represent SD of the mean.