ESEV PBM impairs activation of IRF3 during infection.

<p>A). A549 cells were infected at an m.o.i. of 1 with an H3N2 influenza A virus that expresses either an H6N6 NS1protein with the wt ESEV PBM or mutant ESEA PBM virus. Cell lysates were prepared at the 6 hours post-infection and phosphorylated and total IRF3 levels were quantified in an immunoblot. Densitometry was performed with ImageJ software and the pIRF3 levels were normalized to total IRF3. B. A549 cells were infected with indicated viruses and at 6 hours post-infection cell lysates were prepared and separated into soluble and insoluble fractions as described in Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041251#s4" target="_blank">Methods</a>; levels of viral NP and NS1 proteins in cell fractions were quantified with ImageJ software and normalized to β-actin levels in the corresponding sample. C) A549 cells were infected an m.o.i. of 1 with an H3N2 influenza A virus that expresses either an H6N6 NS1protein with the wt ESEV PBM or mutant ESEA PBM virus. Percentages of cells with nuclear localized-IRF3 were quantified by manually counting as described in Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041251#s4" target="_blank">Methods</a> for three independent experiments. A minimum 200 cells were examined to quantify IRF3 nuclear localization. Error bars represent the standard error of the mean. Statistical difference in effects of NS1 plasmids was determined by student t-test in all the experiments. D). A representative immunofluorescence images used for quantitation in panel C is shown.</p

Effects of NS1 ESEA PBM on activation of IRF3 and IFN-β pre-mRNA levels during influenza A infection.

<p>A) A549 cells were transfected with IFN-β Luciferase and Renilla Luciferase plasmids. After 24 hours, cells were infected with wt or ESEA virus at an m.o.i. of 1.0. Samples were collected at the indicated times for Luciferase assays. IFN-β luciferase values were normalized to Renilla Luciferase. Error bars represent the standard error of the mean. B) A549 cells were infected with either wt or ESEA virus at an m.o.i. of 1.0. Total RNA was isolated at the indicated times post-infection and RT-PCR assays were performed for IFN-β pre-mRNA as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041251#s4" target="_blank">Methods</a>. IFN-β pre-mRNA levels were normalized to GAPDH mRNA levels. Error bars represent the standard error of the mean.</p

ESEV PBM confers interaction between NS1 and MAGI-1.

<p>293T cells were transfected with HA-MAGI-1 and either FLAG-H6N6 NS1 ESEA or FLAG-H6N6 NS1 ESEV plasmids. At 48 hours post-transfection cell extracts were prepared and used in immunoprecipitations with anti-HA or anti-FLAG antibody. Products of immunoprecipitations were examined in immunoblots.</p

Depletion of MAGI-1 activates IRF3.

<p>A) A549 cells were transfected with the indicated siRNAs against MAGI-1, Dlg-1, Scribble, and control siRNAs; lanes labeled “Cells” are non-transfected control cells. After 48 hours, cells extracted were prepared and the levels of indicated proteins were examined in immunoblots. The levels of proteins were measured by densitometry analysis using ImageJ software; the signal for each PDZ protein in non-transfected control cells was arbitrarily assigned a value of 1.0 and PDZ protein levels in transfected cells is shown relative to this 1.0 value. B) A549 cells were transfected with the indicated siRNAs. After 24 hours, cells were processed for immunofluorescence for total IRF3 as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041251#s4" target="_blank">Methods</a> for three independent experiments. Percentages of cells with nuclear localized-IRF3 were quantified by manually counting of at least 200 cells (a representative image for this assay is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041251#pone-0041251-g007" target="_blank">Figure 7C</a>). Error bars represent the standard error of the mean from three independent experiments, with each experiment containing duplicate samples.</p

ESEV PBM with intact CPSF30 binding site impairs NS1 inhibition of IFN-β promoter activation.

<p>A) Cultures of A549 cells were transfected in duplicate with indicated amounts of NS1 expression plasmids (containing intact CPSF30 binding site), IFN-β promoter Luciferase plasmid, and Renilla Luciferase plasmid. At 24 hours post-transfection, cells were re-transfected with poly(I:C) and Luciferase expression was measured 20 hours later. Luciferase expression from the IFN-β promoter plasmid was normalized to protein concentration. Error bars represent the standard error of the mean. B) Cultures of A549 cells were co-transfected in duplicate with indicated amounts of NS1 expression plasmids, IFN-β promoter Luciferase plasmid, Renilla Luciferase plasmids and, full-length RIG-I expression plasmid. Luciferase expression from the IFN-β promoter plasmid was normalized to protein concentration. Error bars represent the standard error of the mean. Statistical difference in effects of NS1 plasmids was determined by student t-test in all the experiments. C) A549 cells were transfected with wt and mutant ESEA NS1 plasmid with intact CPSF30 site. After 48 hours, cells were lysed and analyzed for expression of NS1 in an immunoblots. The NS1 protein levels were normalized to corresponding β-actin level. Densitometry analysis was performed by ImageJ software.</p

ESEV PBM impairs NS1 inhibition of phosphorylation and nuclear localization of IRF3.

<p>A). A549 cells were transfected with wt (ESEV) or PBM mutant (ESEA) NS1 expression plasmids. Cells were re-transfected with poly(I:C) 24 hours later, cell extracts were prepared after 24 hours and levels of phosphorylated IRF3 were evaluated in an immunoblot. Mock-transfected cells were also analyzed. Densitometry was performed with ImageJ software. Values shown are normalized to corresponding total IRF3 levels. B) Percentages of cells with nuclear localized-IRF3 were quantified in three independent experiments by manually counting as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041251#s4" target="_blank">Materials and Methods</a> and panel C below. A minimum 200 cells were examined in each experiment to quantify IRF3 nuclear localization. Error bars represent the standard error of the mean. Statistical difference in effects of NS1 plasmids was determined by student <i>t-test</i> in all the experiments. C). A549 cells were transfected with a wt or mutant ESEA NS1 expression plasmid (with intact CPSF30 binding site). After 24 hours of transfection, cells were activated by transfection of poly(I:C). After 20 hours of activation, cells were processed for immunofluorescence. Nuclei were stained with DAPI; IRF3 is shown as red, and NS1 is shown as green. A representative immunofluorescence images is shown from three independent experiments.</p

ESEV PBM impairs NS1 inhibition of IFN-β promoter activation.

<p>A) Cultures of A549 cells were transfected in duplicate with indicated amounts of NS1 expression plasmids (containing inactivated CPSF30 binding site), IFN-β promoter Luciferase plasmid, and Renilla Luciferase plasmid. At 24 hours post-transfection, cells were re-transfected with poly(I:C) and Luciferase expression was measured 20 hours later. Luciferase expression from the IFN-β promoter plasmid was normalized to Renilla Luciferase expression. Error bars represent the standard error of the mean from three independent experiments, with each experiment containing duplicate samples. B) Cultures of A549 cells were co-transfected in duplicate with indicated amounts of NS1 expression plasmids (containing inactivated CPSF30 binding site), IFN-β promoter Luciferase plasmid, Renilla Luciferase plasmids and, full-length RIG-I expression plasmid. Luciferase expression from the IFN-β promoter plasmid was normalized to Renilla Luciferase expression. Error bars represent the standard error of the mean from three independent experiments, with each experiment containing duplicate samples. Statistical differences in effects of NS1 plasmids were determined by student <i>t-test</i> in all the experiments. C) Cultures of A549 cells were transfected in duplicate with indicated amounts of NS1 expression plasmids (containing intact CPSF30 binding site). At 24 hours post-transfection, cells were re-transfected with poly(I:C) and 24 hours later total RNA isolated and RT-PCR assays were performed for IFN-β pre-mRNA as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041251#s4" target="_blank">Methods</a>. IFN-β pre-mRNA levels were normalized to GAPDH mRNA levels. Error bars represent the standard error of the mean from three independent experiments, with each experiment containing duplicate samples. D) A549 cells were transfected with 500 ng of wt or ESEA mutant NS1 expression plasmids (containing inactivated CPSF30 binding site). Cell extracts were prepared 48 hours later and expression levels of the Flag-tagged NS1 proteins were evaluated in an immunoblot. Densitometry analysis was performed by using ImageJ software. Values shown were normalized to corresponding internal control β-actin protein.</p

NS1 with ESEV PBM co-localizes with MAGI-I and Scribble in cytoplasmic puncta.

<p>A) A549 cells were infected with wt or ESEA mutant virus at an m.o.i. of 1. Cells were processed for immunofluorescence at 24 hours post-infection. Arrows indicate perinuclear puncta where ESEV NS1 and MAGI-1 co-localize. B) Same image as panel A; NS1 is shown as green, MAGI-1 is shown as red, and Scribble is shown as red. Arrows indicate puncta containing co-localization of NS1, MAGI-1, and Scribble.</p

Histology of pleural tumors and cytology of MPE from the mice in NS group.

<p>(A) Hematoxylin-eosin staining of parietal pleura from MPE model (Section ×200) indicated that pleural tumors consisted of adenocarcinomatous cells. (B) Hematoxylin-eosin staining of tumor on the pleural surface from MPE model (Section ×200). (C) Wright’s-Giemsa stain of cells from pleural effusion of MPE model showed LLC cells with large nuclei and visible nucleoli (arrow). MPE: malignant pleural effusion.</p

Immunohistochemistry staining of VEGF-C expression in the pleural tumors.

<p>Positive immunohistochemistry staining of VEGF-C was shown as brown part in each figure. Expression of VEGF-C was accessed by the percentage of positive carcinoma cells and the staining intensity. The positive staining of VEGF-C in NS group (A) and L-ES group (B) indicated high expression of VEGF-C in these groups. Low expression of VEGF-C was shown in Bevacizumab group (C) and H-ES group (D). The expression of VEGF-C was significantly decreased in H-ES group compared with that in NS group or L-ES group or Bevacizumab group.Columns: mean value of each group, bars: ±SD. ***P<0.001, **P<0.01, *P<0.05. ns: no significant difference.</p