FIG 1.

MV-Edm-induced autophagy and autophagic flux enhance oncolytic activity of the virus in NSCLC cells. (A) Translocation of EGFP-MAP1LC3B toward autophagosomes was followed by fluorescence microscopy in A549 cells transiently transfected with a plasmid encoding EGFP-MAP1LC3B grown for 2 h in the presence or absence of MV-Edm (MOI = 0.5). Bars represent 10 μm. The number of EGFP-MAP1LC3B-positive vesicles per cell was quantified by fluorescence microscopy. Bright punctuate structures are GFP⁺ vesicles, indicating autophagosomes. (B) A549 lung cancer cells were transiently transfected with a plasmid encoding EGFP-MAP1LC3B for 24 h followed by infection with MV-Edm (MOI = 0.5) for 6 and 24 h or were left uninfected and cultured in completed medium or in Dulbecco's PBS (DPBS) for 4 h. Cells were then stained for measles virus H protein. Aggregation of EGFP-MAP1LC3B at autophagosomes (green dots) and expression of measles virus H protein (red dots) were evaluated by fluorescence confocal microscopy. Scale bars represent 10 μm. (C) Levels of lipidated MAP1LC3B (LC3-II) 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 (upper panels). The LC3-II/GAPDH ratio was quantified by densitometric analysis (lower panels). (D) MAP1LC3B lipidation was analyzed and quantified in A549 and H1299 cells infected with MV-Edm and grown with or without chloroquine. (E) Degradation of SQSTM1 was monitored by immunoblotting of A549 and H1299 cells after infection by MV-Edm (MOI = 0.2) at 24, 48, and 72 h. (F and G) A549 and H1299 cells transfected with siRNA targeting ATG5, ATG7, or BECN1 or with nontargeting control siRNA (F) or with ATG5 or ATG7 expression plasmids or a mock plasmid (G) for 24 h were infected with MV-Edm (MOI = 0.2) for 48 h. Cell death was quantified using trypan blue staining. Knockdown efficiency for ATG5, ATG7, and BECN1 was monitored at the protein level by Western blotting (F, upper panels). One experiment representative of three (for F) or of two (for G) is shown. Results are means of triplicates. (H) Uninfected A549 and H1299 cells were transfected with plasmids encoding ATG5 or ATG7 or with a mock plasmid for 19 h. Cells were then grown in the presence or absence of chloroquine (CQ) (20 μM) for another 5 h. LC3-II was evaluated by Western blotting. The LC3-II/GAPDH ratio was quantified by densitometry. *, P < 0.05; **, P < 0.01.