Fig. 3

MyD88 inhibition decreases ETECK88 invasion in EGCs through the promotion of autophagy. A MyD88 expression was measured by qRT‒PCR. The EGCs were treated with or without baclofen and then infected with or without ETECK88. B, C EGCs were treated with or without baclofen, followed by WB analysis to test the protein expression of Beclin 1 after ETECK88 infection. D–H MyD88 in EGCs was silenced, and the mRNA expression levels of IL-1β, IL-6, TGF-β, Beclin 1, and LC3 were analyzed using qRT‒PCR after ETECK88 infection. I, J EGCs with or without MyD88 silencing were infected with ETECK88, and then, WB was used to analyze the protein level of Beclin 1. K, L Autophagosomes (yellow) and autolysosomes (red) were counted from at least 20 random cells. M After pCMV-mCherry-GFP-LC3B transfection, EGCs were loaded with or without TJ-M2010-5 and then challenged with or without ETECK88. Confocal microscopic analysis of LC3B. Scale bar, 20 μm. N EGCs were transfected with pEGFP-LC3B, exposed to TJ-M2010-5, and then infected with ETETK88-mCherry. The colocalization of ETECK88-mCherry and autophagosomes was examined by confocal microscopy. The binding regions of bacteria and autophagosomes are denoted by white arrows. Scale bar, 20 μm. O, P EGCs were treated with or without shRNA against the MyD88 plasmid, after which the intracellular survival of ETECK88 was estimated. For qRT‒PCR, GAPDH was used for normalization, and the mean fold changes compared to those in the CON group were calculated according to the 2^−ΔΔCT method. For WB, expression levels were normalized to the expression of β-actin. The data are shown as the mean ± SEM. ns - not significant, * or # p < 0.05, ** p < 0.01, *** p < 0.001