Figure 1

DHAV-1 infection induced autophagy in DEFs. A DEFs were or were not infected with 0.01–10 MOI DHAV-1, as described in Materials and methods. Samples were harvested from three biological replicates at the indicated times, and LC3, VP3 and β-actin protein expression was detected by western blot with specific antibodies. B Relative expression levels of targeted proteins were quantified and analysed. Data represent the mean ± SD of three independent experiments. Differences between two groups were analysed using Student’s t test and are presented as P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***). C DEFs were or were not infected with 1 MOI DHAV-1 for 12 h, 24 h or 48 h and harvested to detect expression of targeted proteins by western blot with specific antibodies. Three biological replicates were performed and tested. D Protein expression ratios of LC3-II/β-actin were quantified and analysed. Data represent the mean ± SD of three independent experiments. Differences between two groups were analysed using Student’s t test and are presented as P < 0.05 (*) and P < 0.001 (***). E DEFs were or were not infected with 1 MOI DHAV-1 for 48 h and observed by TEM. The blue arrows indicate single- or double-membrane autophagosome-like vesicles. F DEFs transfected with GFP-LC3 plasmid were mock infected or infected with DHAV-1 in the absence or presence of 100 nmol/L RAPA, and GFP-LC3 puncta were observed by fluorescence microscopy. Blue, DAPI staining of nuclei; green, GFP-LC3 puncta. Scale bar, 25 μm. G The number of LC3-positive puncta per cell was calculated and is presented as the mean ± SD of three replicates per group. Differences between two groups were analysed using Student’s t test and are presented as P < 0.001 (***). H The relative transcription level of the ATG5 gene was detected by RT–qPCR in the absence or presence of NBS, CQ (50 μmol/L) or DHAV-1 (MOI = 1) for the indicated times, with three replicates per group. RAPA, rapamycin; CQ, chloroquine.