FIG 6.

SQSTM1 mediates MV-Edm-induced mitophagy, contributing to impaired DDX58/IFIH1/MAVS signaling. (A) Mitochondria were fractionated from A549 cells that were not infected or that were infected by MV-Edm at an MOI of 0.2 for 24, 48, and 72 h. Mitochondrion-associated and cytoplasmic SQSTM1 was detected by immunoblotting. COX4I1 and GAPDH were used as loading controls for mitochondrial and cytoplasmic samples, respectively. One representative blot from two independent experiments is shown. (B) Colocalization of autophagosomes and mitochondria was quantified in A549 cells transfected with SQSTM1 siRNA for 24 h followed by transient transfection with pEGFP-Map1lc3b for another 24 h. Cells were then infected with MV-Edm (MOI = 0.5) for 12 h and stained with MitoTracker red before being subjected to confocal microscopy (left panel). 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 panel). Means are shown (n = 30 for each group). (C) Mitochondrial mass was measured by cytometry in MitoTracker green-stained A549 cells transfected with siRNA targeting SQSTM1 or nonspecific control siRNA and cultured in the presence or absence of MV-Edm (MOI = 0.5) for 48 h. 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. (D) The mitochondrial HSPD1 protein level was determined by Western blotting in lysates obtained from A549 lung cancer cells transfected with siRNA targeting SQSTM1 or nonspecific control siRNA and cultured in the presence or absence of MV-Edm (MOI = 0.5) for 48 h. A representative result from two independent experiments is shown. (E) Expression levels of DDX58, IFIH1, and MAVS were determined by immunoblotting in cell lysates from A549 and H1299 cells transfected with siRNA targeting SQSTM1 or with a nontargeting siRNA followed by infection with MV-Edm (MOI = 0.2) for 48 h. Similar results were obtained in two independent experiments. (F) Cell death was quantified by trypan blue exclusion in A549 and H1299 cells transfected with siRNA targeting SQSTM1 or with nontargeting control siRNA for 24 h and cultured in the presence or absence of MV-Edm (MOI = 0.2) for another 48 h. Means + SD for triplicates are shown; *, P < 0.05; **, P < 0.01. Similar results were obtained in three independent experiments.