Figure 5

SpaC induces pyroptosis in zebrafish ZF4 cells rather than in human Caco‐2 cells. (A) Cells were treated with increasing concentrations of SpaC (0, 5, 10 and 20 μg/mL) for 24 h. (B) The flow cytometry results of ZF4 cells treated with recombinant SpaC protein in different dose for 24 h. (C) The flow cytometry results of Caco‐2 cells treated with recombinant SpaC protein in different dose for 24 h. (D) A representative western blot analysis showing GSDMEa and GSDMEb cleavage in ZF4 cells. (E) A representative western blot analysis showing GSDME cleavage in Caco‐2 cells. (F) The relative mRNA expression of genes encoding TLR2, TLR4ba and TLR4bb in ZF4 cells (TLR2: F_1,4 = 0.024; TLR4ba: F_1,4 = 5.550; TLR4bb: F_1,4 = 0.062; n = 3). (G) The relative mRNA expression of genes encoding TLR2 and TLR4 in Caco‐2 cells (TLR2: F_1,4 = 12.387; TLR4: F_1,4 = 0.953; n = 3). (H) Plasmid expressing zTLR4ba or zTLR4baEC‐hTLRIC was transfected into Caco2 cells. (I) The flow cytometry results of Caco‐2 cells or Caco‐2 cells overexpressed zTLR4ba or zTLR4baEC‐hTLRIC treated with 10 μg/mL SpaC protein for 24 h. (J) The strength of interaction (binding affinity) between purified SpaC and TLR proteins was measured by MST technique (n = 3). The Numbers of biologically independent samples are labeled on the violin plots. Box represents median ± interquartile range, whiskers represent 1.5× interquartile range. Statistics: Student's t‐test. p < 0.05, *. MST, MicroScale Thermophoresis; ZF4, zebrafish fibroblast cells; zTLR4ba, zebrafish TLR4ba; zTLR4baEC‐hTLRIC, the fusion of the extracellular domain of zTLR4ba and the transmembrane and intracellular domain of hTLR4.