Figure 7.

V. harveyi evades innate immunity through synergistic inhibition of MyD88 by eIF3k and ATG5.A and B, overexpression of eIF3k induces ATG5. MIC cells were transfected the indicated plasmids for 36 h. Endogenous proteins were detected by immunoblot (A) and ATG5 mRNAs was detected by qPCR (B). C, MyD88 induces ATG5 in a dose-dependent manner. EPC cells were transfected with the indicated plasmids for 48 h followed by immunoblot. D, ATG5 mRNAs was induced by V. harveyi. Expression of ATG5 and MyD88 in MIC was determined by qPCR after V. harveyi stimulation at for different times. E, ATG5 enhanced the degradation of MyD88 by eIF3k. EPC cells were transfected with the indicated plasmids for 24 h before LPS stimulation. F, ATG5 increases the adhesion of V. harveyi to MKC cells. Cells were transfected with ATG5, vector was used for control. Lysates from V. harveyi infected cells were incubated on LB plates for 12 h, and the cfu was counted. G and H, MIC cells were transfected with ATG5-si and eIF3k-si. Cells were treatment with LPS for 6 h at 48 h post-transfection. The expression MyD88 were detected by qPCR (G), autophagic flux was analysis by immunoblot (H). I, knocking down ATG5 weakened the adhesion of V. harveyi to MKC cells. Methods were same to (F). J and M, HeLa cells were transfected with the indicated plasmids for 30 h, then stained with FITC (green) or Cy3 (red). The nuclei were stained by DAPI (blue). Pictures were taken by FCFM. Scale bar, 20 μm; original magnification ×40. K, L, and N, HEK293 cells were transfected with the indicated plasmids for 32 h before Co-IP and followed by immunoblot. O, a schematic presentation of interactions among MyD88, eIF3k and ATG5. The data are shown as the mean ± SD of three independent experiments. (∗) p < 0.05, (∗∗) p< 0.01 versus the controls. eIF3k, eukaryotic translation initiation factor 3k; EPC, epithelioma papulosum cyprini; MIC, M. miiuy intestine cell; MKC, M. miiuy kidney cell.