FIG 5.

MV-Edm induces mitophagy leading to mitochondrial degradation. (A) Levels of ATG5-ATG12 conjugates were assessed by Western blotting in lysates obtained from A549 and H1299 lung cancer cells infected with MV-Edm at an MOI of 0.5 or left uninfected for 6, 9, and 24 h. A representative result from two independent experiments is shown. (B) Colocalization of autophagosomes and mitochondria was quantified in A549 cells transiently transfected with pBABEpuro-EGFP-Map1lc3b and infected by MV-Edm (MOI = 0.5) for 4, 12, and 24 h or left uninfected. Cells were then stained with MitoTracker stain and subjected to confocal microscopy (left panels). Scale bars = 10 μm. Colocalization (yellow dots) of mitochondria (red) with autophagosomes (green puncta) was quantified by calculating Pearson's correlation coefficient [PCC, R(r)] (right panels). Means are shown (n = 30 for each time point). **, P < 0.01; N.S, not significant. (C) Subcellular analysis of A549 cells infected without (left panel) or with (middle and right panels) MV-Edm (MOI = 1; 24 h) was performed by electron microscopy. Arrowheads depict double-layer structures that contain mitochondria in an MV-Edm-infected cell. Scale bar = 2 μm (left), 5 μm (middle), or 1 μm (right). (D) Mitochondrial mass was measured by cytometry in MitoTracker green-stained A549 cells 12, 24, and 48 h after infection with MV-Edm (MOI = 0.5). An overlay of histograms representative of 3 independent experiments (left panel), and quantification of mitochondrial mass as mean fluorescence intensity averaged from 3 independent experiments (right panel) are shown. (E) Mitochondrial HSPD1 protein level was determined by Western blotting in lysates obtained from A549 lung cancer cells infected with MV-Edm at an MOI of 0.5 for 12, 24, and 48 h or left uninfected. A representative result from two independent experiments is shown.