Autophagy accompanied by autophagosome and amphisome formation was induced in the brain tissues of EV71 MP4-infected suckling mice. Seven-day-old ICR suckling mice were inoculated intracranially with EV71 MP4 (5 × 105 pfu/mouse). Mice were sacrificed at 24 hr p.i.. (A) Autophagosome and amphisome formation was detected in the brain stem of the EV71 MP4-infected suckling mice. The tissue sections were treated with either mouse monoclonal or rabbit polyclonal anti-LC3 antibody and anti-MPR rabbit polyclonal antibody anti-EV71 VP1 mouse monoclonal antibody. Green: MPR; Red: EV71 VP1; Yellow: colocalization of MPR and EV71 VP1. Arrow indicates colocalization. (B) Seven-day-old ICR suckling mice (n = 3, each group) were pretreated with 3-MA (0.3 mg/mouse) 2 hr before inoculation of EV71 MP4 (5 × 105 pfu/mouse). Mice brain tissues were harvested and total protein lysate was collected after EV71 infection for 6 hr, 12 hr and 24 hr. The total protein lysate from each group was pooled together and the expression levels of EV71 VP1, LC3-II, and BECN1 were measured by Western blotting using specific antibodies. β-actin was used as the internal control. The numbers under each band are the quantification of the band intensity. For the comparison of VP1 protein expression levels, the intensity of EV71 infected cells without 3-MA treatment at 6 hr p.i. was set as 1 (normalized with the intensity of β-actin). For the comparison of LC3-II and BECN1 levels, the intensity of Sham PBS control cells without 3-MA treatment at 24 hr p.i. was set as 1 (normalized with the intensity of β-actin).