Figure 3

SpaC and LPS induce pyroptosis in gut microbiota‐independent and ‐dependent manners. (A) GF zebrafish (4‐dpf) were immersed with 10 μg/mL SpaC or LPS for 3 d. (B) A representative western blot analysis showing Caspase‐3 activation, GSDMEa cleavage, Caspy2 activation and GSDMEb cleavage in GF zebrafish. (C) The casp3a^−/− zebrafish (4‐dpf) were immersed with 10 μg/mL SpaC or LPS for Day 3. (D) A representative western blot analysis showing GSDMEa cleavage and GSDMEb cleavage in casp3a^−/− zebrafish. (E) Gut microbiota of zebrafish fed with SpaC or LPS diet was transferred to GF zebrafish for Day 3. (F) A representative western blot analysis showing Caspy2 activation and GSDMEb cleavage in GF zebrafish received the gut microbiota from one‐month old zebrafish fed the SpaC1.0 or LPS0.2 diet for 3 weeks. (G) Zebrafish (4‐dpf) with caspy2 knockdown were immersed with 10 μg/mL SpaC or LPS for Day 3. (H) A representative western blot analysis showing GSDMEa cleavage and GSDMEb cleavage in caspy2 knockdown zebrafish. (I) ZF4 cells were treated with 10 μg/mL SpaC or LPS when gene encoding GSDMEa or GSDMEb were silenced. (J) Representative plots of cell pyroptotic/apoptotic rate. (K) Statistical pyroptotic rates at 24 h posttreatment (Scrambled siRNA: F_2,27 = 4.954; gsdmea: F_2,27 = 4.067; gsdmeb: F_2,27 = 2.449; n = 10). Box represents median ± interquartile range, whiskers represent 1.5× interquartile range in the violin plots. Statistics: Student's t‐test. Treatments in plots labeled with different letters on top represent statistically significant results (p < 0.05). dpf, day postfertilization; GF, germ‐free; siRNA, specific small interfering RNA.