Establishment of our drug screening model.

<p>(A) Schematic representation of the drug screening model. Beclin1 plays several important roles in autophagy, the dissociation of Beclin1 from Bcl2 is essential for autophagy. Autophagy either supports IAV replication or induces autophagic cell death which may result in acute lung injury (left). Bcl2 binds with Beclin1 to form Beclin1-Bcl2 heterodimer and inhibits autophagy (right). Beclin1-binding proteins (such as MyD88, TRIF and HMGB1), Bcl2-binding proteins (such as BNIP3, Bad, Noxa, Puma, BimEL and Bik) and the activations of ERK1/2, JNK1, IKK, TLRs and DAPK signal pathways under oxidative stress, endoplasmic reticulum (ER) stress and energy stress can promote the dissociation of Beclin1 from Bcl2, and finally promote autophagy. IAV infection can elevate the expressions of many aforementioned proteins and activate aforementioned signal pathways. Drugs inhibiting oxidative stress, ER stress, energy stress, and the activations of ERK1/2, JNK1, IKK, TLRs and DAPK signal pathways may inhibit the dissociation of Beclin1 from Bcl2, sequentially inhibit autophagy, and finally impair IAV replication or inhibit autophagic cell death and acute lung injury. In our study, Beclin1 and Bcl2 were fused with C’- and N’- fragments of a red fluorescence protein (RFP), respectively. The amino acid sequence of the linker was RPACKIPNDLKQKVMNH. After cotransfection, the intact RFP would reconstitute through the interaction of Beclin1 and Bcl2. The fluorescence intensity (FI) of the reconstituted RFP, which was determined at 610 nm after excitation at 587 nm using a microplate reader (Tecan infinite M1000), represents the level of the Beclin1-Bcl2 heterodimer, which is positively relevant with the degree of autophagy inhibition and antiviral activity. (B) Reconstitution of RFP. A549 cells were transfected or cotransfected with pMN-Bcl2 and pMC-Beclin1, after 8 h, only cotransfected cells appeared a lot of red fluorescence. (C) The influence of HMGB1 and MyD88 on the Beclin1-Bcl2 heterodimer. Beclin1-binding proteins HMGB1 and MyD88 were expected to disrupt the Beclin1-Bcl2 heterodimer, after cotransfection as the graph indicated, the FI was really significantly decreased, and the cells of same batch were subjected to the co-immunoprecipitation (co-IP) assay (right), the results satisfied our expectation. (D) The influence of ERK1/2 inhibitor (U0126, 10 µM), ERK1/2 activator (EGF, 100 ng/ml), JNK/p38 inhibitor (SB203580, 40 µM), p38 MAPK activator (anisomycin, 10 µM), antioxidant (NAC, 2 mM) and oxidant (H2O2, 100 µM) on the dissociation of Beclin1-Bcl2 heterodimer. After cotransfection, A549 cells were treated with these inhibitors and activators, after 8 h, the FI was measured, the inhibitors (U0126, SB203580) and antioxidant (NAC) could significantly elevate the FI, which meant that they could inhibit the dissociation of Beclin1 from Bcl2, as comparing with their corresponding activators (EGF, anisomycin) and oxidant (H₂O₂), the cells of the same batch were also subjected to the co-IP assay (right), the results also satisfied our expectation. Normal rabbit IgG was used as a control in co-IP assay. Data shown were the mean ± SD of three independent experiments and shown as the fold change to the corresponding control. *<i>P</i><0.05, **<i>P</i><0.01.