Figure 7.

Delayed autophagosome formation by BEFV is suppressed by 5-aminoimidazole-4-carboxamide-1-β-riboside (AICAR) or aspirin. (A) MDBK cells were infected with BEFV at an MOI of 1 with or without pretreatment of AICAR (1 mM) and aspirin (5 mM), respectively. Cell lysates were collected at the indicated time points and subjected to immunoblotting with the respective antibodies. The levels of indicated proteins in the mock group were considered onefold. The activation and inactivation folds indicated below each lane were normalized against values for the mock control group. β‐actin was used for the internal control for normalization. All experiments were conducted in three independent experiments. Signals in all Western blots were quantified with ImageJ software. The predicted size of each protein was labeled at the right-hand side in kDa. (B) Induction of autophagy in MDBK cells expressing the mCherry-GFP-LC3 protein was monitored after pretreatment with AICAR or aspirin, respectively, for 30 min and then infected with BEFV at an MOI of 1 for 18 h. LC3 fluorescent puncta were observed using fluorescence microscopy. Cell nuclei were stained with DAPI. Scale bars, 25 mm. (C) The numbers of LC3 punta were calculated from the results from panel (B). Significance between the treatments was determined by Duncan’s Multiple Range Test (MDRT) using SPSS software (Version 20.0). Means with common alphabets (a, b, c) denotes no significance at p <0.05. Each value represents mean ± SE of three independent experiments.