Fig. 8: Ben_0691 upregulates host cell IRG-47 through the ATP5A1-AMPK-mTOR pathway.

A EBL cells were transfected with pCAGGS-Myc or ATP5A1-Myc for 12 h, and then transfected with siNC or siATP5A1-1 for 48 h. Cell lysates were collected to analyze the levels of P-AMPK, AMPK, P-mTOR, mTOR, IRG-47, ATP5A1 and β-actin by Western blotting. B EBL cells were incubated with AICAR (100 μM) or RAPA (1 μg/ml) for 12 h, and harvested for Western blotting to analyze the phosphorylation levels of AMPK (P-AMPK/AMPK,) or mTOR (P-mTOR/mTOR). C, D The selected concentrations of AICAR or RAPA had no effect on cell viability in EBL. E, F EBL cells were transfected with pCAGGS-Myc or ATP5A1-Myc for 36 h, and then incubated with AICAR or RAPA for 12 h. Cell lysates were collected for Western blotting to assay the levels of P-AMPK, AMPK, P-mTOR, mTOR, IRG-47, ATP5A1, and β-actin. G EBL cells were transfected with pEGFP-C1 or Ben_0691-GFP for 12 h, and then transfected with siNC or siATP5A1-1 for 48 h. Cell lysates were subjected for Western blotting of the levels of P-AMPK, AMPK, P-mTOR, mTOR, IRG-47, ATP5A1 and β-actin. H EBL cells were transfected with pCAGGS-Myc or ATP5A1-Myc, followed by incubation with or without AICAR or RAPA. The cells were harvested for qPCR to detect IFN-γ mRNA level. All data are presented as the means ± SDs and were normalized to the corresponding values in control cells. Significance was assessed by a two-tailed Student’s t test (C, D, n  =  6 independent experiments) or by one-way ANOVA with Tukey’s multiple comparison test (H, n  =  3 independent experiments). *p < 0.05.