|
Fig. 1
Caspase-5-like activity is essential for pyroptosis in zebrafish fibroblasts. a ZF4 zebrafish fibroblasts were infected with wild-type (EIB202) or 0909I E. piscicida for 2 h at a multiplicity of infection (MOI) of 50, or left uninfected. Supernatants from the indicated ZF4 cells were analyzed for cell death, as measured by lactate dehydrogenase (LDH) release. b ZF4 cells were infected with 0909I E. piscicida for 2 h at an MOI of 50. Relative caspase activity was then measured by incubating cell lysates with fluorogenic and chromogenic substrates of caspase-1 (YVAD), caspase-2 (VDQQD), caspase-3/7 (DEVD), caspase-4 (LEVD), caspase-5 (WEHD), caspase-8 (IETD), and caspase-9 (LEHD). c, d ZF4 cells were treated with caspase-3/7, pan-caspase, caspase-1, caspase-4, and caspase-5 inhibitors (Ac-DEVD-CHO, Z-VAD-FMK, Z-YVAD-FMK, Ac-LEVD-CHO, and Z-WEHD-FMK, respectively). c LDH release for cell death was measured 2 h after 0909I E. piscicida infection. d Images were taken after 0909I E. piscicida infection for 2 h. Propidium iodide (PI) was added to detect the loss of plasma membrane integrity. Arrows indicate cells exhibiting pyroptotic-like features. Scale bar, 50 µm. e–g ZF4 cells were primed with Pam3CSK4 for 4 h, before being stimulated with cholera toxin B subunit (CTB) plus LPS, LPS, or CTB alone for 12 h. e Supernatants from the indicated ZF4 cells were analyzed for cell death, as measured by lactate dehydrogenase (LDH) release. f Cytosolic LPS-delivered ZF4 cells were treated with indicated caspase inhibitors as in Fig. 1c, Supernatants from the indicated ZF4 cells were analyzed for cell death, as measured by lactate dehydrogenase (LDH) release. g Relative caspases activity was measured by incubating cell lysates with indicated fluorogenic and chromogenic substrates as in Fig. 1b. a–g Results are representative of at least three independent experiments, and error bars denote the SD of triplicate wells. *p < 0.05 (t test)