The interaction between NOLC1 and IAV NS1 protein promotes host cell apoptosis and reduces virus replication

Figures

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  Figure 1. NS1 interacts with NOLC1 (A) NS1 and NOLC1 interaction determined by pull-down assay. Lane 1, Lysate from 293T cells expressing flag-NOLC1 was directly subjected to western blot analysis using anti-FLAG anitbody. Lane 2, Lysate from 293T cells expressing flag-NOLC1 that was incubated with His-tagged NS1-MagneHis™ beads and then eluted. Lane 3, Lysate from 293T cells expressing flag-NOLC1 that was incubated with MagneHis™ beads without adsorbed His-tagged NS1. (B) Interaction between NS1 and NOLC1 as determined by coimmunoprecipitation. Flag-NS1(H3N2) and NOLC1 were co-expressed in the 293T cells, the cell lysate was subjected to immunoprecipitation using anti-FLAG antibody, and the coimmunoprecipitated proteins were detected by western blot with the anti-NOLC1 antibody.

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  Figure 2. NS1 inhibits the synthesis of NOLC1 293T cells were cultured and transfected with p3×Flag-CMV-14-NS1(H3N2). The levels of NS1 and NOLC1 were detected at 48 h post-transfection by western blot analysis. GAPDH was used as an internal reference.

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  Figure 3. Screening of NOLC1-specific shRNA (A) Fluorescence detection of the RNAi. A549 cells were co-transfected with pDsRed-N1-NOLC1 and four different NOLC1 shRNA plasmids: pGPU6/GFP/Neo-sh667 (c); pGPU6/GFP/Neo-sh1262 (d); pGPU6/GFP/Neo-sh1376 (e); and pGPU6/GFP/Neo-sh1775 (f). After 48 h, each culture was observed and photographed with a confocal laser scanning microscope in order to compare the efficacy of the gene-silencing effect. A549 cells transfected with pDsRed-N1-NOLC1 was used as a blank (a) and A549 cells co-transfected with pGPU6/GFP/Neo-shNC and pEGFP-N1-NS1 were used as a negative control (b). (B) The effect of RNAi on NOLC1 expression as determined by western blot analysis. A549 cells were transfected with pGPU6/GFP/Neo-sh1775 plasmid and NOLC1 expression was analyzed by immunoblot analysis at 48 h post-transfection. A549 cells transfected with pGPU6/GFP/Neo-shNC plasmid was used as the negative control. (C) The staining intensity on the western blots were quantified using Totallab 2.0 software and statistically analyzed using SPSS V13.0. Values are the mean ± SD of three independent experiments performed in triplicate.

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  Figure 4. Double staining and flow cytometry analysis of cell apoptosis A549 cells transfected with different combinations of pGPU6/GFP/Neo-sh1775, pEGFP-N1-NS1, and pDsRed-N1-NOLC1 were double stained with Annexin V and PI, and the apoptosis index was determined by flow cytometry. Percent apoptosis for each of the sample-types are presented as the mean ± standard error from three separate experiments. The levels of apoptosis in transfected cell types in e – g were significantly different (p < 0.01) than the levels in the non-transfected and transfected cell types a – d. (P <0.01). (a) Cells without transfection; (b) cells transfected with pEGFP-N1; (c) cells transfected with pDsRed-N1; (d) cells transfected with pGPU6/GFP/Neo; (e) cells transfected with pEGFP-N1-NS1; (f) cells transfected with pGPU6/GFP/Neo-sh1775; (g) cells co-transfected with pEGFP-N1-NS1 and pGPU6/GFP/Neo-sh1775; (h) cells transfected with pEGFP-N1-NS1 and pDsRed-N1-NOLC1.