Relationship between viability and IFN-α production by pDCs. (A) Purified pDCs were cultured with 10nM bortezomib for 2 hours and 0.5μM ODN2216 was added. The cells were harvested after 4 hours, stained with FITC-conjugated annexin V, and analyzed for viability by flow cytometry (top panel). IFN-α mRNA was quantitated by real-time RT-PCR, and the expression levels were normalized to those of GUS (bottom panel). The data are normalized to the value obtained with ODN2216 in the absence of bortezomib. *P < .05. The data are shown as means ± SE of 3 independent experiments. (B) After purified pDCs (1 × 10⁵/mL) were cultured without or with 50μM Z-VAD-FMK for 1 hour, the indicated concentrations of bortezomib were added. After 6 hours, 0.5μM ODN2216 was added and the cells and supernatants were harvested 18 hours later. The cells were stained with FITC-conjugated annexin V and analyzed by flow cytometry. The percentages of cell death specifically induced by bortezomib were calculated (top panel). Concentrations of IFN-α in the supernatants were measured by ELISA (bottom panel). The data are normalized to the value obtained without bortezomib. *P < .05; **P < .01; NS, not significant. The data are shown as means ± SE of 6 independent experiments.