Figure 4

LGG SpaC pilin triggers intestinal pyroptosis by interacting with zebrafish TLR4ba. (A) One‐month old zebrafish were immersed with recombinant SpaC at concentrations of 10 μg/mL for Day 7. The relative mRNA expression of genes encoding TLR2, TLR4ba and TLR4bb in zebrafish intestine as measured by qRT‐PCR (TLR2: F_1,8 = 0.116; TLR4ba: F_1,10 = 18.075; TLR4bb: F_1,6 = 0.912; n = 6). (B) Characterization of the interaction between SpaC and zebrafish TLR4ba by BiFC. (C) The TLR4ba was knocked down by using morpholino oligonucleotides in zebrafish larvae. (D) A representative western blot analysis showing GSDMEa and GSDMEb cleavage in zebrafish larvae with TLR4ba knockdown upon SpaC immersion. 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 < 0.01, **. BiFC, bimolecular fluorescent complementation; qRT‐PCR, quantitative real‐time PCR reaction; TLR, toll‐like receptor; Negative Ctrl (nontransfection), mock (cotransfection of pBiFC‐VC155 and pBiFC‐VN173), positive Ctrl (cotransfection of pBiFC‐bFosVC155 and pBiFC‐bjunVN173), experimental group (cotransfection of pBiFC‐VN173‐SpaC and pBiFC‐VC155‐zTLR4ba).