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. 2018 Dec 14;9:3120. doi: 10.3389/fmicb.2018.03120

FIGURE 2.

FIGURE 2

NS1 suppresses the autophagosome formation through both the dsRNA-binding activity and the activation of PI3K-Akt signaling pathway. (A–D) HeLa cells (A) and A549 cells (C) were infected with either Wt, R38AK41A, or Y89F virus at MOI of 3. At 10 h post-infection, cells were subjected to the indirect immunofluorescence assays with anti-LC3 antibody (green). Nuclei were stained with DAPI (blue). Scale bar, 10 μm. The average percentage of cells exhibiting LC3 puncta relative to total infected cells and standard deviations determined from three independent experiments were shown in panel (B) (HeLa cells) and (D) (A549 cells) (AP; Autophagosome, n > 100). The statistical significance was determined by Student’s t-test, ∗∗∗P < 0.001. (E) HeLa cells were infected with Wt, R38AK41A, or Y89F virus at MOI of 3. At 20 h post-infection, the cell lysates were prepared and subjected to western blotting assays with anti-LC3B, anti-NP, and anti-β-actin antibodies. (F) HeLa cells were infected with Wt, R38AK41A or Y89F virus at MOI of 3. At 4 h post-infection, the cell lysates were prepared and subjected to western blotting assays with anti-phospho-Akt, anti-Akt, anti-phospho-TSC2, anti-TSC2, anti-NP, and anti-β-actin antibodies.