Figure 5.

eIF3k mediates MyD88 degradation through the autophagy pathway.A, 3-MA and NH₄Cl blocked eIF3k-mediated MyD88 degradation. EPC cells were transfected with the indicated plasmids for 36 h, cells were stimulated with MG132 (10 μM), 3-MA (10 mM), or NH₄Cl (20 mM) for an additional 8 h, DMSO was as a negative control. The level of MyD88 was detected and normalized to Tubulin. B, CHX was jointed use with DMSO or 3-MA to stimulate the EPC cells at 36 h post-transfection. MyD88 level was detected and normalized to Tubulin. C, effects of 3-MA on LPS-induced degradation of MyD88. EPC cells were transfected with eIF3k and MyD88 for 24 h, then cells were pre-treated with 3-MA for 2 h, followed by LPS stimulation for the indicated times. D, NH₄Cl blocked eIF3k-mediated MyD88 degradation in a dose-dependent manner. EPC cells were cotransfected with the indicated plasmids for 36 h, NH₄Cl was added for an additional 8 h. E, EPC cells were cotransfected with MyD88 and LC3-GFP, together with eIF3k or control vector. The puncta of LC3-GFP were detected by fluorescence microscope. Scale bar, 100 μm. F, effects of LPS and V. harveyi treatment on autophagy. MIC cells were treated with LPS and V. harveyi for 6 h at 24 h posttransfection, and then subjected for fluorescence microscopy. Scale bar, 20 μm; original magnification ×40. LC3-GFP dots were quantified using the Image J software. A minimum of 50 cells were scored for each condition. G, activated autophagy increases eIF3k-mediated MyD88 degradation. EPC cells were treated with LPS at the indicated time at 24 h posttransfected with eIF3k and MyD88. H, effects of EBSS treatment on autophagic flux. HeLa cells were cotransfected with MyD88, eIF3k, and LC3-GFP-RFP for 24 h, and then cells were treated with EBSS for 2 h. The nuclei were stained by DAPI (blue), pictures were taken by FCFM. Scale bar, 20 μm; original magnification ×40. I, eIF3k enhance the autophagy in MIC. MIC was transfected with the indicated plasmids for 48 h before immunoblot. J, eIF3k enhanced the association between MyD88 and LC3B. EPC cells were transfected with the indicated plasmids for 36 h before Co-IP and immunoblot. K, effects of eIF3k on the conversion of LC3B-I to LC3B-II. EPC cells were transfected with the indicated plasmids for 48 h before immunoblot. L, eIF3k plays a role in the prophase of autophagy. EPC cells were co-transfected with MyD88 and eIF3k or control vector for 36 h, cells were stimulated with 3-MA (10 mM) and CQ (50 mM) for an additional 8 h. The level of LC3B was detected and normalized to Tubulin. The data are shown as the mean ± SD of three independent experiments. (∗) p< 0.05, (∗∗) p< 0.01 versus the controls. 3-MA, 3-methyladenine; eIF3k, eukaryotic translation initiation factor 3k; EPC, epithelioma papulosum cyprini; MIC, M. miiuy intestine cell.