Journal: PLoS Pathogens

Article Title: DENV NS1 and MMP-9 cooperate to induce vascular leakage by altering endothelial cell adhesion and tight junction

doi: 10.1371/journal.ppat.1008603

Figure Lengend Snippet: ( A ) HEK293T cells were co-transfected with HA-MMP-9 and Flag-Cap, Flag-M, Flag-Prm, Flag-E, Flag-NS1, Flag-NS2A, Flag-NS2B, Flag-NS3, Flag-NS4A, or Flag-NS4B. Cell lysates were immunoprecipitataed using anti-Flag antibody, and analyzed using anti-Flag and anti-HA antibody. Cell lysates (40 μg) was used as Input. ( B ) HEK293T cells were co-transfected with HA-MMP9 and Flag-NS1, Cell lysates were immunoprecipitataed using anti-HA antibody, and analyzed using anti-Flag and anti-HA antibody. Cell lysates (40 μg) was used as Input. ( C, D ) HEK293T cells were co-transfected with empty vector or VC-155-MMP-9 and VN173-NS1/E/NS4A. At 24 h post-transfection, living cells were observed by confocal microscopy. The quantification of YFP-positive cells was determined by ImageJ software (D). ND means not detection. ( E ) Purified His-SARS-CoV-2-N (5 μg) or His-DENV2-NS1 (5 μg) was incubated with purified no-tagged MMP-9 protein (3 μg) for 24 h, Mixtures were incubated with Ni-NTA Agarose beads. Mixtures were analyzed by immunoblotting using anti-MMP9, anti-NS1, anti-His antibody. Untreated protein including His-SARS-CoV-2-N (1 μg), His-DENV2-NS1 (1 μg), or no-tagged MMP-9 protein (1 μg) were analyzed by immunoblotting using anti-MMP9, anti-NS1, and anti-His antibody (as input). ( F ) Yeast strain AH109 were co-transformed with combination of binding domain (BD-p53, BD-MMP-9, and BD-Lam) and activation domain (AD-T, AD-NS1) plasmid. Transfected yeast cells were grown on SD-minus Trp/Leu double dropout plates, and colonies were replicated on to SD-minus Trp/Leu/Ade/His fourth dropout plates to check for the expression of reporter genes. ( G, H ) Schematic diagram of wild-type MMP-9 protein and truncated mutants MMP-9 protein (D1 to D9) (G). HEK293T cells were co-transfected with HA-NS1 and Flag-MMP-9 truncated mutants (D1 to D9). Cell lysates were immunoprecipitated using anti-Flag antibody, and analyzed using anti-Flag and anti-HA antibody. Cell lysates (40 μg) was used as Input (H). Dates were representative of three independent experiments.

Article Snippet: Commercial purified DENV2-NS1 were purchased from Sino Biological (40243-V07H) and Commercial purified MMP-9 protein were purchased from RD company (P14780).

Techniques: Transfection, Plasmid Preparation, Confocal Microscopy, Software, Purification, Incubation, Western Blot, Transformation Assay, Binding Assay, Activation Assay, Expressing, Immunoprecipitation

Journal: PLoS Pathogens

Article Title: DENV NS1 and MMP-9 cooperate to induce vascular leakage by altering endothelial cell adhesion and tight junction

doi: 10.1371/journal.ppat.1008603

Figure Lengend Snippet: ( A ) PMA-differentiated THP-1 macrophages (top) or HUVEC cells (bottom) were treated with infectious or UV- inactivated DENV2 at MOI = 5 for 48h. Intracellular DENV2 E RNA (bottom) was determined by qRT-PCR analysis. Mock: untreated cells. Control: supernatant of C6/36 cells without DENV2 infection. ( B and C ) PMA-differentiated THP-1 macrophages were infected with DENV2 for different times at MOI = 5 (B) or at different MOI for 24 h (C). Intracellular MMP-9 RNA (top) and DENV2 E RNA (bottom) was determined by qRT-PCR analysis, MMP-9 proteinase activity in the supernatants was determined by gelatin zymography assays and proteins in cell extract (middle) were analyzed by Western blotting. ( D and E ) HUVEC cells were infected with DENV2 for different times at MOI = 5 (D C ) and at different MOI for 24 h (E). Intracellular MMP-9 RNA (top) and DENV2 E RNA (bottom) was determined by qRT-PCR analysis, MMP-9 proteinase activity in the supernatants was determined by gelatin zymography assays and proteins in cell extract (middle) were analyzed by Western blotting. ( F ) HUVEC cells or PMA-differentiated THP-1 macrophages were infected with DENV2 at MOI = 5 for 24 h. Viral copies were quantified by RT-PCR. ( G ) HUVEC cells or PMA-differentiated THP-1 macrophages were equally distributed to four 12-hole plates and infected with DENV2 at MOI = 5 for 24 h. MMP-9 protein in cell supernatants were measured by ELISA (top) and indicated proteins in cell extract were analyzed by WB (bottom). Dates were representative of three independent experiments. ns means not significant. Values are mean ± SEM, P ≤0.05 (*), P ≤0.01 (**), P ≤0.001 (***).

Article Snippet: Commercial purified DENV2-NS1 were purchased from Sino Biological (40243-V07H) and Commercial purified MMP-9 protein were purchased from RD company (P14780).

Techniques: Quantitative RT-PCR, Infection, Activity Assay, Zymography, Western Blot, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: PLoS Pathogens

Article Title: DENV NS1 and MMP-9 cooperate to induce vascular leakage by altering endothelial cell adhesion and tight junction

doi: 10.1371/journal.ppat.1008603

Figure Lengend Snippet: ( A ) PMA-differentiated THP-1 macrophages were infected with DENV2 for different times at MOI = 5, NS1 protein in Supernatants were analyzed by ELISA (top). Cell lysates were analyzed by immunoblotting (bottom). ( B ) Confluent monolayers of HUVEC cells were grown on polycarbonate membrane system and treated with the supernatants came from DENV2 infected HUVEC cells or THP-1 cells for 24 h or pre-incubated with 600nM SB-3CT (a specific inhibitor of MMP-9 protein) or 600 nM SC75741 for 1h. Endothelial permeability was evaluated by measuring trans-endothelial electrical resistance (TEER) (ohm) using EVOM2 epithelial voltohmmeter. ( C – I ) IFNAR -/- C57BL/6 mice were intravenously injected with 300 μl DENV2 at a dose of 1×10 6 PFU/mouse (n = 6), pre-treated with 300 μl PBS containing MMP-9 specific inhibitor SB-3CT (5 mg/kg per mice) by intraperitoneal injection for 90 min and then treated with DENV2 (1×10 6 PFU/mouse), repeat treated with SB-3CT (5 mg/kg per mice) on the fourth day after DENV2 (NGC) infection (n = 6), or 300 μl PBS containing the same volume DMSO as a control group (n = 4). 7 days after infection, mice were euthanasia, and the tissues were collected. MMP-9 RNA in the blood was determined by qRT-PCR (upper) and MMP-9 protein in the serum was measured by ELISA (lower). Points represent the value of each serum samples (C). Evans blue dye was intravenously injected into mice 7 days after DENV infected groups (n = 5), control groups (n = 4) and DENV+SB-3CT (n = 5) (C–E). The dye was allowed to circulate for 2 hours before mice were euthanasia, tissues include liver (D), spleen (E) and lung (F) were collected, and the value of Evans blue was measured at OD 610 . Histopathology analysis of tissues includes Liver (G), Spleen (H) and Lung (I) after DENV infection. ( J ) Monolayers of HUVEC cells grown on Transwell inserts were incubated for 48 h with MMP-9 protein (100 ng/ml) or NS1 protein (5 μg/ml) or NS1 (5 μg/ml) plus different concentration of MMP-9 (50 ng/ml to 100ng/ml) or pre-treated with 600 nM SB-3CT or 600 nM SC7574 for 1 h, then incubated with NS1 plus MMP-9. The TEER (ohm) was measured at indicated time points. Dates were representative of two to three independent experiments. ns means not significant. Values are mean ± SEM, P ≤0.05 (*), P ≤0.01 (**), P ≤0.001 (***).

Article Snippet: Commercial purified DENV2-NS1 were purchased from Sino Biological (40243-V07H) and Commercial purified MMP-9 protein were purchased from RD company (P14780).

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Western Blot, Incubation, Permeability, Injection, Quantitative RT-PCR, Histopathology, Concentration Assay

Journal: PLoS Pathogens

Article Title: DENV NS1 and MMP-9 cooperate to induce vascular leakage by altering endothelial cell adhesion and tight junction

doi: 10.1371/journal.ppat.1008603

Figure Lengend Snippet: ( A – F ) IFNAR -/- C57BL/6 mice were intravenously injected with 300 μl DENV2 at a dose of 1×10 6 PFU/mouse (n = 6), pre-treated with 300 μl PBS containing MMP-9 specific inhibitor SB-3CT (5 mg/kg per mice) by intraperitoneal injection for 90 min and then treated with DENV2 (1×10 6 PFU/mouse), repeat treated with SB-3CT (5 mg/kg per mice) on the fourth day after DENV2 (NGC) infection (n = 6), or 300 μl PBS containing the same volume DMSO as a control group (n = 4). 7 days after infection, mice were euthanasia, and the tissues were collected. The indicated proteins in Lung (A), spleen (B) and Liver (C) were measured by Western-blot. The expression of β-catenin in Liver (D), spleen (E), and Lung (F) by Immunohistochemistry analysis. ( G ) HUVEC cells were respectively transfected with plasmid encoding MMP-9 (2 μg) or NS1 (2 μg) or NS1 (1 ug) plus MMP-9 (1 μg) for 24 h or firstly co-transfected with plasmid encoding NS1 (1ug) plus MMP-9 (1 μg) for 12 h, then treated with 600nM SB-3CT for 12 h. The indicated proteins in cell extract were analyzed by WB. ( H–K ) HUVEC cells were treated with NS1 protein (5 μg/ml) or MMP-9 protein (100 ng/ml) or NS1 (5 μg/ml) plus MMP-9 (100 ng/ml) or pre-incubated with 600 nM SB-3CT for 1 h, then treated with NS1 (5 μg/ml) plus MMP-9 (100 ng/ml) for 6 h, The distribution of endogenous β-catenin (H) or ZO-1 (J) protein were visualized under confocal microscope. The quantifications of relative β-catenin (I) or ZO-1 (K) intensities were determined by ImageJ software. The quantification of protein was used by ImageJ software (A–G). (D–F) +++ means Percentage contribution of high positive cells; ++ means Percentage contribution of positive cells; + means Percentage contribution of low positive cells;—means Percentage contribution of negative cells. ns means not significant. All dates were representative of two to three independent experiments.

Article Snippet: Commercial purified DENV2-NS1 were purchased from Sino Biological (40243-V07H) and Commercial purified MMP-9 protein were purchased from RD company (P14780).

Techniques: Injection, Infection, Western Blot, Expressing, Immunohistochemistry, Transfection, Plasmid Preparation, Incubation, Microscopy, Software

Journal: PLoS Pathogens

Article Title: DENV NS1 and MMP-9 cooperate to induce vascular leakage by altering endothelial cell adhesion and tight junction

doi: 10.1371/journal.ppat.1008603

Figure Lengend Snippet: ( A ) HEK293T cells were transfected with plasmid encoding HA-NS1 plus Flag-β-catenin. Cell lysates were immunoprecipitated using anti-HA antibody, and analyzed using anti-Flag, anti-HA, or anti-β-catenin antibody. Cell lysates (40 μg) were used as Inputs. ( B ) Hela cells were transfected with plasmid encoding HA-NS1, Cell lysates were immunoprecipitated using anti-HA antibody, and analyzed using anti-HA or anti-β-catenin antibody. Cell lysates (40 μg) was used as Input. ( C , D ) HEK293T cells (C) or Hela cells (D) were co-transfected with plasmid encoding HA-NS1 plus Flag-ZO-1, Cell lysates were immunoprecipitated using anti-HA antibody, and analyzed using anti-Flag or anti-HA antibody. Cell lysates (40 μg) were used as Inputs. ( E , F ) HEK293T cells were transfected with Flag-β-catenin (E) or Flag-ZO-1 (F) and incubated with purified His-SARS-CoV-2-N (5 μg) or His-DENV2-NS1 (5 μg) for 24 h, cell extracts were incubated with Ni-NTA Agarose beads. Mixtures were analyzed by immunoblotting using anti-β-catenin, anti-NS1, anti-His, anti-ZO-1 antibody. Untreated proteins including His-SARS-CoV-2-N (1 μg) and His-DENV2-NS1 (1 μg) and HEK293T cell lysates were analyzed by immunoblotting using anti-β-catenin, anti-NS1, anti-His, and anti-ZO-1 antibody (as input). ( G , H ) Hela cells were co-transfected with plasmid encoding HA-NS1 plus Flag-MMP-9, Cell lysates were immunoprecipitated using anti-Flag (G) or anti-β-catenin antibody (H), and analyzed using anti-Flag, anti-HA or anti-β-catenin antibody. Cell lysates (40 μg) was used as Input. ( I, J ) Hela cells were co-transfected with plasmid encoding HA-NS1, HA-MMP-9, and Flag-ZO-1 (I) or Flag-NS1, HA-MMP-9, and Flag-ZO-1 (J). Cell lysates were immunoprecipitated using anti-Flag (top) or anti-HA antibody (bottom), and analyzed using anti-Flag, anti-HA or anti-MMP-9 antibody. Cell lysates (40 μg) was used as Input. ( K , L ) HUVEC cells were treated with NS1 protein (5 μg/ml), MMP-9 protein (100 ng/ml), and NS1 protein (5 μg/ml) plus MMP-9 protein (100 ng/ml), respectively, for 6 h. The distributions of the membrane marker (Dil) (yellow), the endogenous β-catenin (yellow) and ZO-1 (yellow) proteins and the extracellular NS1 (red) and MMP-9 (green) proteins were visualized under confocal microscope. The quantifications of co-localization fluorescence were determined by ImageJ software (L). ND means not detected. All dates were representative of three independent experiments.

Article Snippet: Commercial purified DENV2-NS1 were purchased from Sino Biological (40243-V07H) and Commercial purified MMP-9 protein were purchased from RD company (P14780).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Incubation, Purification, Western Blot, Marker, Microscopy, Fluorescence, Software

Journal: PLoS Pathogens

Article Title: DENV NS1 and MMP-9 cooperate to induce vascular leakage by altering endothelial cell adhesion and tight junction

doi: 10.1371/journal.ppat.1008603

Figure Lengend Snippet: C57BL/6 mice and MMP-9 -/- mice were injected intravenously DENV2 NS1 protein [10 mg/kg (n = 5)], the same volume of PBS was also tail vein injected to C57BL/6 mice and MMP-9 -/ - mice (n = 5) as control group. Another group of MMP-9 -/- mice (n = 5) were injected intravenously DENV2 NS1 protein (10 mg/kg) plus recombinant mouse MMP-9 protein (70 μg /kg). ( A – C ) After 24 h post-injection, mice were intravenously injected with Evans blue dye. The dye was allowed to circulate for 2h before mice were euthanized, and tissue include Lung (A), spleen (B), and Liver (C) were collected. The value of Evans blue was measured at OD 610 . ( D – I ) After 24 h post-injection, mice were euthanized and tissue were collected. The indicated proteins in Lung (D), spleen (E), and Liver (F) were measured by Western-blot. The expression of β-catenin in Lung (G), spleen (H), and Liver (I) were analyzed by Immunohistochemistry. All dates were representative of two to three independent experiments. The quantification of protein was used by ImageJ software (D–I). (G–H) +++ means Percentage contribution of high positive cells; ++ means Percentage contribution of positive cells; + means Percentage contribution of low positive cells;—means Percentage contribution of negative cells. ns means not significant. Values are mean ± SEM, P ≤0.05 (*), P ≤0.01 (**), P ≤0.001 (***).

Article Snippet: Commercial purified DENV2-NS1 were purchased from Sino Biological (40243-V07H) and Commercial purified MMP-9 protein were purchased from RD company (P14780).

Techniques: Injection, Recombinant, Western Blot, Expressing, Immunohistochemistry, Software

Journal: PLoS Pathogens

Article Title: DENV NS1 and MMP-9 cooperate to induce vascular leakage by altering endothelial cell adhesion and tight junction

doi: 10.1371/journal.ppat.1008603

Figure Lengend Snippet: DENV non-structural protein 1 (NS1) induces MMP-9 expression through activating the nuclear factor κB (NF-κB) signaling pathway. Additionally, NS1 interacts with MMP-9 and facilitates the enzyme to alter the adhesion and tight junctions and vascular leakage in human endothelial cells and mice tissues. Moreover, NS1 recruits MMP-9 to interact with β-catenin and Zona occludens protein-1/2 to degrade the important adhesion and tight junction proteins, thereby inducing endothelial hyperpermeability and vascular leakage in human endothelial cells and mice tissues.

Article Snippet: Commercial purified DENV2-NS1 were purchased from Sino Biological (40243-V07H) and Commercial purified MMP-9 protein were purchased from RD company (P14780).

Techniques: Expressing