Figure 2. rVP1 induced BECN1-independent, and ATG5- and ATG7-dependent autophagy. BECN1 stable knockdown RAW264.7 cell line, RAWsh-Becn-1, was generated by shRNA silencing as described in Materials and Methods. (A) Knockdown of BECN1 decreased serum starvation-mediated but not rVP1-induced LC3 lipidation. RAW264.7 cells stably knocked down with scrambled or Becn1 shRNA were pretreated with or without 2 μM CQ for 30 min and then incubated with or without serum starvation for 160 min or 4 μM rVP1 for 4 h as indicated. Cells were lysed and analyzed by immunoblotting using antibodies against BECN1, SQSTM1 and LC3, respectively. ACTB was used as a loading control. (B) rVP1 induced formation of double-membrane autophagosomes independent of BECN1. RAW264.7 cells stably knocked down with scrambled or Becn1 shRNA were serum-depleted for 160 min or incubated with 4 μM rVP1 for 4 h as indicated. After treatment, cells were collected and observed with transmission electron microscopy. Data represent means ± SEM of volume fraction of autophagic compartments; **p < 0.01., N.S., not significant. (C) rVP1 did not induce LC3 lipidation after knockdown of ATG5 and ATG7. RAW264.7 cells transfected with scrambled or siRNA of Atg5 and Atg7 were pretreated with 2 μM CQ and then incubated with or without 4 μM rVP1 for 4 h as indicated. Cell lysates were collected and analyzed by immunoblot using anti-LC3, anti-ATG5 and anti-ATG7 antibodies. ACTB was used as a loading control. Blots are representative of three independent experiments.