Fig. 2
GABABR activation protects EGCs from ETECK88 invasion by enhancing autophagy. A ELISA was used to measure the levels of GABABR in the intestines of the mice. B EGCs were subjected to qRT‒PCR to determine the mRNA levels of GABABR. C, D The levels of GABABR in EGCs were analyzed by WB. E–H The mRNA expression levels of IL-6, TGF-β, Beclin 1, and LC3 in EGCs were assessed by qRT‒PCR. Cells were treated with or without baclofen and then infected with or without ETECK88. I, J EGCs were pretreated with or without baclofen and then analyzed for Beclin 1 protein expression after ETEC infection. K, L Average number of autophagosomes (yellow) and autolysosomes (red) in each cell (>20 cells/group). M Transiently transfected EGCs expressing pCMV-mCherry-GFP-LC3B were treated with or without baclofen and then challenged with or without ETECK88. Confocal microscopic analysis of LC3B. Scale bar, 20 μm. N Confocal microscopic analysis of the interaction between ETECK88-mCherry and autophagosomes. EGCs were transfected with the pEGFP-LC3B plasmid, and the cells were treated with or without baclofen and then infected with or without ETECK88-mCherry. The binding regions of bacteria and autophagosomes are denoted by white arrows. Scale bar, 20 μm. O, P Intracellular survival of ETECK88-treated EGCs pretreated with or without baclofen. 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. * or # p < 0.05, ** or ## p < 0.01, *** or ### p < 0.001