DV infection activated caspase-1 and induces pyroptosis. (A) GM-Mφ or M-Mφ was infected with DV (MOI = 5 and 30) as described in “DV infection to macrophages.” At 48 hours after infection, cells were stained with trypan blue and observed under a light microscope. Bar represents 100 μm. (B) LDH released from DV-infected GM-Mφ. The amounts of LDH released in the supernatants were determined as described in “LDH release assay.” Values represent mean ± SEM of triplicate independent experiments. **P < .01; ***P < .001 (Student t tests). (C) TUNEL assay. DNA damage in DV-infected GM-Mφ was determined by TUNEL assay as described in “TUNEL assay.” M1 indicates percentage of TUNEL-positive cells. (D) The amounts of caspase-1 p20 released from mock or DV-infected GM-Mφ were determined by ELISA. (E-F) Inhibition of DV-induced IL-18 production (E) and cell death (F) by caspase-1 inhibitor. GM-Mφ were preincubated with DMSO, caspase-1 inhibitor (50μM), and caspase-3 inhibitor (50μM), and then infected with DV (MOI = 30) as described in “Blocking assay.” At 48 hours after infection, supernatants were subjected to ELISA assay to determine IL-18 levels and LDH amounts. Data are the mean ± SEM from 3 independent experiments. *P < .05; **P < .01; ***P < .001 (Student t tests). DV5, 30 indicates MOI = 5 and 30, respectively; and ND, not detectable.