Journal: bioRxiv

Article Title: Virus-host interactome reveals host cellular pathways perturbed by tick-borne encephalitis virus infection

doi: 10.1101/2025.02.26.640292

Figure Lengend Snippet: a. Schematic view of TBEV viral proteins. b. Comprehensive interactome analysis was conducted to study interactions between TBEV and host proteins. c. Viral baits expressed in HEK293T cells and analyzed by immunoblot assay using anti-Flag antibody. d, e. Enrichment analysis of TBEV-interacted host proteins (d) and the proteins (e) significantly deregulated by TBEV infection. f. UpSet plot showing the overlap of flavivirus-host protein–protein interactions from other studies.( , ) Each bar shows the interactions shared by only the marked studies at the bottom.

Article Snippet: Antibodies against HA, Flag, GAPDH, Actin, CDK1, CDK2, CDK4, CDK6, p16, p21, p53, Cyclin B1, TBEV-NS1, as well as CoraLite488- conjugated goat anti-mouse IgG antibody were obtained from Proteintech (Wuhan, China).

Techniques: Western Blot, Infection, Protein-Protein interactions

Journal: bioRxiv

Article Title: Virus-host interactome reveals host cellular pathways perturbed by tick-borne encephalitis virus infection

doi: 10.1101/2025.02.26.640292

Figure Lengend Snippet: a. An integrated protein-protein interaction network showed high-confidence TBEV-host interactions. Viral proteins are represented as rhombus nodes. Edge intensity reflects the MIST value associated with the interaction. Solid thick edges represent the most significant interactions, while light edges correspond to low MIST (Molecular Interaction Search Tool) value (0.5 < MIST < 0.99). b-f. Validation of virus-host interactions using co-immunoprecipitation. Flag-tagged empty vector (EV) or viral proteins were overexpressed in HEK293T cells, and CHUK co-precipitated with prM ( b ), SYVN1 co-precipitated with NS2B ( c ), SPCS1 co-precipitated with NS4A ( d ), endogenous Ephrin B1(EFNB1) co-precipitated with NS4B ( e ), along with PAF1 and LEO1 co-precipitated with NS5( f ), were detected using flag beads and immunoblot assay.

Article Snippet: Antibodies against HA, Flag, GAPDH, Actin, CDK1, CDK2, CDK4, CDK6, p16, p21, p53, Cyclin B1, TBEV-NS1, as well as CoraLite488- conjugated goat anti-mouse IgG antibody were obtained from Proteintech (Wuhan, China).

Techniques: Biomarker Discovery, Virus, Immunoprecipitation, Plasmid Preparation, Western Blot

Journal: bioRxiv

Article Title: Virus-host interactome reveals host cellular pathways perturbed by tick-borne encephalitis virus infection

doi: 10.1101/2025.02.26.640292

Figure Lengend Snippet: a. The schematic illustration outlines the network diffusion analysis process. The proteome results were initially integrated with the virus-host interactome, and subsequently mapped to the global human protein-protein interactome to identify affected host pathways. b. Subnetworks derived from network diffusion predict the correlation of TBEV NS5 with spliceosome (| Log 2 FC|>0.1, P value < 0.05). c. Subnetworks resulting from network diffusion depict the correlation of TBEV prM with factors involved in autophagy (|Log 2 FC|>0.1, P value < 0.05). d, e. Additional subnetworks derived from network diffusion predict the correlation of TBEV NS5 with DNA damage response ( d ) and cell cycle ( e ) (|Log 2 FC|>0.1, P value < 0.05). Black edges denote connections present in ReactomeFI. The thickness of the line reflects the combined score provided by STRING. The thickness of directed edges is proportional to the random-walk transition probability.

Article Snippet: Antibodies against HA, Flag, GAPDH, Actin, CDK1, CDK2, CDK4, CDK6, p16, p21, p53, Cyclin B1, TBEV-NS1, as well as CoraLite488- conjugated goat anti-mouse IgG antibody were obtained from Proteintech (Wuhan, China).

Techniques: Diffusion-based Assay, Virus, Derivative Assay

Journal: bioRxiv

Article Title: Virus-host interactome reveals host cellular pathways perturbed by tick-borne encephalitis virus infection

doi: 10.1101/2025.02.26.640292

Figure Lengend Snippet: a. Vero cells were either mock-infected or infected with TBEV at various MOI (0.1, 1.0 and 10), cells were collected at 48 hours post infection (hpi) and their distribution was analyzed by flow cytometry. b. The experiments in ( a ) were repeated for three times, and the proportions of cells at different cell cycle phases were shown in column graph. c. Vero cells were mock-infected or infected with TBEV at the MOI of 1.0, cells were collected at 12, 18, 24, 36 and 48 hpi, and the percentages of cells in G0/G1 ( up ), S ( middle ) and G2/M ( down ) phase were analyzed by flow cytometry. d-i . Vero cells were synchronized to G0/G1 ( d ), S ( f ) and G2/M ( h ) phases via no-serum starvation, adding 0.85mM Thymi or 25 ng/mL Noco for 20 h. Subsequently, the cells were either mock-infected or infected with TBEV at an MOI of 10, cells were collected and analyzed using flow cytometry. The experiments were repeated for three times, and the proportion of cells in were shown in column graphs. j-l. Vero cells were synchronized to different stages and subsequently infected with TBEV, the mRNA level of TBEV envelope (E) gene was analyzed at 48 hpi by probe qPCR ( j ). The protein level of TBEV NS1 was analyzed at 48 hpi by immunoblot ( k, l ).

Article Snippet: Antibodies against HA, Flag, GAPDH, Actin, CDK1, CDK2, CDK4, CDK6, p16, p21, p53, Cyclin B1, TBEV-NS1, as well as CoraLite488- conjugated goat anti-mouse IgG antibody were obtained from Proteintech (Wuhan, China).

Techniques: Infection, Flow Cytometry, Western Blot

Journal: bioRxiv

Article Title: Virus-host interactome reveals host cellular pathways perturbed by tick-borne encephalitis virus infection

doi: 10.1101/2025.02.26.640292

Figure Lengend Snippet: a. Cells were transfected with the plasmids of Flag-tagged empty vector (EV) and ten TBEV viral proteins, the cell distribution was analyzed by flow cytometry. b. The Flag-EV, 0.5 and 1.0 μg Flag-NS5 were transfected into A549 cells, the cell distribution was analyzed by flow cytometry. The experiment was repeated for three times, and the percentage of cells were shown in column graph. c. Cells were transfected with Flag-EV or Flag NS5 together with HA-P300, the interaction between NS5 and P300 was analyzed by co-immunoprecipitation analysis. d. The colocalization of P300 (green) and NS5 (red) were examined by immunofluorescence analysis. Scale bar: 10 μm. e-g . Cells transfected with NS5 were collected after 48 h, the expression of CDK4, CDK6 and P16 was analyzed by immunoblot ( e ) and qPCR analysis ( f ). The relative expression of CDK4, CDK6 and P16 was shown in column graph ( g ). h. A549 cells transfected with HA-EV, HA P300 and P300 Hm were further transfected with NS5, the distribution of cells was analyzed by flow cytometry. The experiments were repeated for three times, and the proportions of cells were shown in column graph, the expression of indicated proteins was assessed by immunoblot assay. i. Cells transfected with P300 siRNA and NC siRNA (siNC) were further transfected with NS5, the distribution of cells was analyzed by flow cytometry, and the proportions of cells and the relative expression of P300 mRNA were shown in column graph. j. Cells transfected with HA-EV, HA P300 and P300 Hm were mock-infected or infected TBEV, the distribution of cells was analyzed by flow cytometry. k. Cells transfected with P300 siRNA and NC siRNA were mock-infected or infected TBEV, the distribution of cells was analyzed by flow cytometry. l. Outline of G0/G1 cell cycle arrest regulated by TBEV NS5.

Article Snippet: Antibodies against HA, Flag, GAPDH, Actin, CDK1, CDK2, CDK4, CDK6, p16, p21, p53, Cyclin B1, TBEV-NS1, as well as CoraLite488- conjugated goat anti-mouse IgG antibody were obtained from Proteintech (Wuhan, China).

Techniques: Transfection, Plasmid Preparation, Flow Cytometry, Immunoprecipitation, Immunofluorescence, Expressing, Western Blot, Infection

Journal: bioRxiv

Article Title: Virus-host interactome reveals host cellular pathways perturbed by tick-borne encephalitis virus infection

doi: 10.1101/2025.02.26.640292

Figure Lengend Snippet: a. Cells were either mock-infected or infected with TBEV, and then collected at 12, 24, 36 and 48 hpi, the expression levels of cell cycle regulators were detected by immunoblot using indicated antibodies. M: mock, I: infection. b. Representative images of brain organoid generation from iPSCs. Scale bar, 500 µm. c. Microscopic images of brain organoids, either mock-infected or infected with TBEV at 0- and 3-days post-infection (dpi). Scale bar, 200 µm. d. Microscopic images depicting 42-day-old iPSC-derived organoids, featuring TUJ1 (neurons), SOX2 (progenitors), Olig2 (oligodendrocytes), and NeuN (neuronal nuclei) markers. Scale bar, 500 µm. e, f . Brain organoids were either mock-infected or infected with TBEV at the MOI of 2.0, the expression of CDK4 ( e ) and P16 ( f ) in brain organoids were examined using immunofluorescence analysis. CDK4 and P16 were visualized in red, TBEV NS1was visualized in green, and the nuclei were stained in blue. Scale bar: 50 μm.

Article Snippet: Antibodies against HA, Flag, GAPDH, Actin, CDK1, CDK2, CDK4, CDK6, p16, p21, p53, Cyclin B1, TBEV-NS1, as well as CoraLite488- conjugated goat anti-mouse IgG antibody were obtained from Proteintech (Wuhan, China).

Techniques: Infection, Expressing, Western Blot, Derivative Assay, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Virus-host interactome reveals host cellular pathways perturbed by tick-borne encephalitis virus infection

doi: 10.1101/2025.02.26.640292

Figure Lengend Snippet: a, b . A549 cells were either mock-infected or infected with TBEV, the cells were then treated with indicated concentration of C646 ( a ) and CPI-637 ( b ), the supernatants were collected at 48 hpi, and the mRNA level of TBEV E gene was analyzed by probe qPCR. c-f . Cells mock-infected or infected with TBEV at the MOI of 10 were treated with 10 μM C646 and CPI-637, the expression of TBEV NS1 protein was detected by immunoblot ( c, d ) and immunofluorescence analysis ( e ), and the cell cycle distribution was analyzed by flow cytometry ( f ). Scale bar: 50 μm. g. Cells transfected with HA-EV, HA P300 and P300Hm were infected TBEV, the mRNA level of TBEV E gene in the supernatants was analyzed by probe qPCR. h. Cells transfected with P300 siRNA and NC siRNA were infected TBEV, the mRNA level of TBEV E gene in the supernatants was analyzed by probe qPCR. i. A549 cells were treated with the indicated concentrations of chrysin for 2 h, the cells were then infected with TBEV, the supernatants were collected at 48 hpi, and the mRNA level of TBEV E gene was analyzed by probe qPCR. j-l. Cells were treated with 10 μM chrysin for 2 h, the cells were then mock-infected or infected with TBEV at the MOI of 10, the expression of TBEV NS1 protein was detected by immunoblot ( j ), and the cell distribution was analyzed by flow cytometry ( k ). The percentage of cells ( l ) upon chrysin treatment was shown in column graphs. Scale bar: 50 μm.

Article Snippet: Antibodies against HA, Flag, GAPDH, Actin, CDK1, CDK2, CDK4, CDK6, p16, p21, p53, Cyclin B1, TBEV-NS1, as well as CoraLite488- conjugated goat anti-mouse IgG antibody were obtained from Proteintech (Wuhan, China).

Techniques: Infection, Concentration Assay, Expressing, Western Blot, Immunofluorescence, Flow Cytometry, Transfection

Journal: bioRxiv

Article Title: Virus-host interactome reveals host cellular pathways perturbed by tick-borne encephalitis virus infection

doi: 10.1101/2025.02.26.640292

Figure Lengend Snippet: a. Virus-protein interaction network and predicted drug targets. Potential drug-protein interactions were predicted by NeDREx using the TrustRank algorithm, integrating data from various biomedical databases and experimentally validated targets. Nodes represent proteins and edges indicate their interactions. b-d. Cells were pretreated with indicated inhibitors targeting RIOK1/BRD9 ( b ), XIAP ( c ) and KAT6A ( d ) before TBEV infection for 48 h. The IC 50 value was determined using probe qPCR. Cell viability was assessed using cell counting (CCK-8) assay in the absence of viral infection. The percentage of viral titer compared with DMSO treatment (red) and cell viability (black) depicted. Error bars represent the mean ± SEM of three biological replicates.

Article Snippet: Antibodies against HA, Flag, GAPDH, Actin, CDK1, CDK2, CDK4, CDK6, p16, p21, p53, Cyclin B1, TBEV-NS1, as well as CoraLite488- conjugated goat anti-mouse IgG antibody were obtained from Proteintech (Wuhan, China).

Techniques: Virus, Infection, Cell Counting, CCK-8 Assay

Journal: Emerging microbes & infections

Article Title: ERp57 facilitates ZIKV-induced DNA damage via NS2B/NS3 complex formation.

doi: 10.1080/22221751.2024.2417864

Figure Lengend Snippet: Figure 5. ERp57 is critical for ZIKA nonstructural protein expression and function. (A) Representative immunofluorescent staining images show the localization of ZIKV NS5 (green). WT RD cells were seeded in 24-well plates with pre-coated cover glasses, then infected with ZIKA virus (MOI=1) for the indicated time. Cells were fixed and stained with anti-NS5 (green) fluorescence antibody and DAPI (blue) and visualized under confocal microscope. (B) and (C) Both wild type RD cells and knockout cells were infected by ZIKV (MOI=1) at the indicated time and protein level was analyzed by western blot. (D) Both WT and KO RD cells were infected with ZIKV (MOI=1) for the indicated time. Cells were fixed and stained with anti-NS1 (green) fluorescence antibody and DAPI (blue) and visualized under confocal microscope. (E) Both wild type RD cells and ERp57-KO cell line were transfected with NS1-flag plasmid or vector, then infected with ZIKV (MOI=1) for 48 h. Protein samples were blotted to investigate the expression level of NS1 and other DDR related factors. (F) Western blot images of WT and ERp57-KO cells transfected with NS1-Flag plasmid or vector for 24 h.

Article Snippet: Plasmids containing genomes encoding ZIKV nonstructural proteins were purchased from Addgene, including NS1 (#79633), NS2B (#79637), NS3 (#79635), NS4a (#79636), NS5 (#79639).

Techniques: Expressing, Staining, Infection, Virus, Fluorescence, Microscopy, Knock-Out, Western Blot, Transfection, Plasmid Preparation

Journal: Journal of Cellular and Molecular Medicine

Article Title: RNF7 promotes glioma growth via the PI3K / AKT signalling axis

doi: 10.1111/jcmm.17656

Figure Lengend Snippet: RNF7 was overexpressed in glioma and negatively associated with prognosis. (A) Heat map shows the top upregulated genes in different groups. (B) WB was used to detect the expression of RNF7 protein in tumour and normal tissues. (C) qRT‐PCR was used to detect RNF7 mRNA levels in tumour tissues and normal tissues. (D, E) IHC staining and scoring were used to detect the protein levels of RNF7 in tumour tissues and normal tissues. (F) Log‐rank test of DFS or OS was conducted. Data were presented as mean ± SD from three independent experiments. ** p < 0.01 and *** p < 0.001

Article Snippet: Primary antibodies were used to detect RNF7 (ab181986; Abcam), AKT (ab179463; Abcam), p‐AKT (ab192623; Abcam), PI3K (ab191606; Abcam) and p‐PI3K (ab182651; Abcam).

Techniques: Expressing, Quantitative RT-PCR, Immunohistochemistry

Journal: Journal of Cellular and Molecular Medicine

Article Title: RNF7 promotes glioma growth via the PI3K / AKT signalling axis

doi: 10.1111/jcmm.17656

Figure Lengend Snippet: Association of RNF7 expression with clinicopathological characteristics in human gliomas

Article Snippet: Primary antibodies were used to detect RNF7 (ab181986; Abcam), AKT (ab179463; Abcam), p‐AKT (ab192623; Abcam), PI3K (ab191606; Abcam) and p‐PI3K (ab182651; Abcam).

Techniques: Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: RNF7 promotes glioma growth via the PI3K / AKT signalling axis

doi: 10.1111/jcmm.17656

Figure Lengend Snippet: Single factor and multiple factor analysis table

Article Snippet: Primary antibodies were used to detect RNF7 (ab181986; Abcam), AKT (ab179463; Abcam), p‐AKT (ab192623; Abcam), PI3K (ab191606; Abcam) and p‐PI3K (ab182651; Abcam).

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: RNF7 promotes glioma growth via the PI3K / AKT signalling axis

doi: 10.1111/jcmm.17656

Figure Lengend Snippet: Overexpression of RNF7 facilitates cell cycle progression and cell growth and inhibits apoptosis. (A) Cell growth curves measured by CCK‐8 between Vector and oeRNF7. (B) RNF7 overexpression facilitated colony formation and histogram quantification (panels). (C) Invasion assays showing that overexpression of RNF7 facilitates cell invasion. The numbers of invading cells are shown. Bars: 50 μm. (D) Typical pictures of live animal imaging between Vector and oeRNF7. Representative pictures of Ki‐67 staining between Vector and oeRNF7. Data were presented as mean ± SD from three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Article Snippet: Primary antibodies were used to detect RNF7 (ab181986; Abcam), AKT (ab179463; Abcam), p‐AKT (ab192623; Abcam), PI3K (ab191606; Abcam) and p‐PI3K (ab182651; Abcam).

Techniques: Over Expression, CCK-8 Assay, Plasmid Preparation, Imaging, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: RNF7 promotes glioma growth via the PI3K / AKT signalling axis

doi: 10.1111/jcmm.17656

Figure Lengend Snippet: Knockdown of RNF7 suppresses cell cycle progression and cell growth and promotes apoptosis. (A) Cell growth curves measured by CCK‐8 assay between si‐NC and siRNF7#2. (B) RNF7 overexpression inhibited colony formation and histogram quantification (panels). (C) Invasion assays showing that RNF7 knockdown inhibits cell invasion. The numbers of invading cells are shown. Bars: 50 μm. (D) Typical pictures of live animal imaging between si‐NC and siRNF7#2. Representative pictures of Ki‐67 staining between si‐NC and siRNF7#2. Data were presented as mean ± SD from three independent experiments. * p < 0.05 and ** p < 0.01

Article Snippet: Primary antibodies were used to detect RNF7 (ab181986; Abcam), AKT (ab179463; Abcam), p‐AKT (ab192623; Abcam), PI3K (ab191606; Abcam) and p‐PI3K (ab182651; Abcam).

Techniques: CCK-8 Assay, Over Expression, Imaging, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: RNF7 promotes glioma growth via the PI3K / AKT signalling axis

doi: 10.1111/jcmm.17656

Figure Lengend Snippet: RNF7 activates the PI3K/AKT signalling and thereby drives tumour growth. (A) Expression of RNF7, MCM7, PCNA, AKT, p‐AKT, PI3K and p‐PI3K was detected by WB between si‐NC, siRNF7#1 and siRNF7#2. (B) Expression of RNF7, MCM7, PCNA, AKT, p‐AKT, PI3K and p‐PI3K was detected by WB between Vector and oeRNF7.

Article Snippet: Primary antibodies were used to detect RNF7 (ab181986; Abcam), AKT (ab179463; Abcam), p‐AKT (ab192623; Abcam), PI3K (ab191606; Abcam) and p‐PI3K (ab182651; Abcam).

Techniques: Expressing, Plasmid Preparation

Journal: Journal of Cellular and Molecular Medicine

Article Title: RNF7 promotes glioma growth via the PI3K / AKT signalling axis

doi: 10.1111/jcmm.17656

Figure Lengend Snippet: RNF7 influences tumour growth via activating the PI3K/AKT signalling. (A) Expression of RNF7, MCM7, PCNA, AKT, p‐AKT, PI3K and p‐PI3K was measured by WB in different treatment groups. (B) Cell growth curves measured by CCK‐8 assay in different treatment groups. (C) Cell growth measured by colony formation assay in different treatment groups and histogram quantification (panels). Data were presented as mean ± SD from three independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001

Article Snippet: Primary antibodies were used to detect RNF7 (ab181986; Abcam), AKT (ab179463; Abcam), p‐AKT (ab192623; Abcam), PI3K (ab191606; Abcam) and p‐PI3K (ab182651; Abcam).

Techniques: Expressing, CCK-8 Assay, Colony Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: RNF7 promotes glioma growth via the PI3K / AKT signalling axis

doi: 10.1111/jcmm.17656

Figure Lengend Snippet: RNF7 influences tumour growth via activating the PI3K/AKT signalling. (A) RNF7 overexpression could promote tumour cell migration, invasion and the effect could be attenuated by treatment with PI3K/AKT signalling pathway inhibitors LY294002. (B) Typical pictures of live animal imaging in different treatment groups and representative pictures of Ki‐67 staining in different treatment groups. Data were presented as mean ± SD from three independent experiments. ** p < 0.01 and *** p < 0.001

Article Snippet: Primary antibodies were used to detect RNF7 (ab181986; Abcam), AKT (ab179463; Abcam), p‐AKT (ab192623; Abcam), PI3K (ab191606; Abcam) and p‐PI3K (ab182651; Abcam).

Techniques: Over Expression, Migration, Imaging, Staining

Journal: FEBS letters

Article Title: Influenza NS1 interacts with p53 and alters its binding to p53-responsive genes, in a promoter-dependent manner.

doi: 10.1016/j.febslet.2013.08.006

Figure Lengend Snippet: Fig. 1. NS1 is involved in the IAV-induced stabilization of p53. (A) Stability assay in IAV-infected cells. A549 cells were mock-infected or infected with influenza virus A/ Moscow/10/99 (H3N2) with an MOI of 2 or 0.1 and analysed at different time-points. Stability was assessed by monitoring relative protein levels (RPL) of p53 during a 1 h time period, post treatment with 50 lM cycloheximide (CHX). Mean values ± standard deviation from three independent experiments are presented. ⁄ and ⁄⁄⁄ for P-value <0.05 and 0.001, respectively. (B) Stability assay in presence of NS1. A549 cells were transfected with either an empty plasmid (pCI-empty) or a plasmid expressing NS1 from the H3N2 strain (pCI-NS1-H3N2), and p53 stability was evaluated 36 h post-transfection. Mean values ± standard deviation from three independent experiments are presented. ⁄P-value <0.05. (C) The efficient expression of NS1, p53 and its targets 36 h post-transfection were analysed by western blot. (D) Analysis of the interaction between NS1 and p53 by co-immunoprecipitation (co-IP). H1299 cells were transfected with either an empty plasmid (pCI-empty) or a plasmid expressing NS1 (pCI-NS1- H3N2), and a plasmid expressing p53 (pSV-p53). After 48 h, cells were scraped into ice-cold PBS and analysed using a co-immunoprecipitation assay with an anti-p53 polyclonal antibody (CMI), or a control IgG.

Article Snippet: A mouse anti-NS1 monoclonal antibody (Santa-Cruz ref sc-130568) was used to control a possible binding of NS1 to promoter regions.

Techniques: Stability Assay, Infection, Virus, Standard Deviation, Transfection, Plasmid Preparation, Expressing, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay, Control

Journal: FEBS letters

Article Title: Influenza NS1 interacts with p53 and alters its binding to p53-responsive genes, in a promoter-dependent manner.

doi: 10.1016/j.febslet.2013.08.006

Figure Lengend Snippet: Fig. 3. NS1 inhibits p53 transcriptional activity. (A) A549 cells were transfected with the pG13-luc reporter plasmid together with increasing concentrations of NS1 expressing plasmid, (pCI-NS1-H3N2, left panel). H1299 cells (p53 null) were then co-transfected with 1 lg of pCI-empty or pCI-NS1-H3N2 and increasing concentrations of p53 expressing plasmid (pSV-p53, right panel). (B) A549 cells were transfected with p21, Bax or Mdm2 luciferase reporter plasmid constructs together with 1 lg of pCI-empty plasmid or pCI-NS1-H3N2 C. H1299 cells were co-transfected with 1 lg of pCI-NS1-H3N2 and 1 lg of each specific promoter reporter construct together with increasing concentrations of pSV-p53. In A. B. and C. Cells were harvested at 36 hpi and RLU were measured. Mean values ± standard deviation from three independent experiments are presented. (ns, P > 0.05 and ⁄⁄⁄,<0.001).

Article Snippet: A mouse anti-NS1 monoclonal antibody (Santa-Cruz ref sc-130568) was used to control a possible binding of NS1 to promoter regions.

Techniques: Activity Assay, Transfection, Plasmid Preparation, Expressing, Luciferase, Construct, Standard Deviation

Journal: FEBS letters

Article Title: Influenza NS1 interacts with p53 and alters its binding to p53-responsive genes, in a promoter-dependent manner.

doi: 10.1016/j.febslet.2013.08.006

Figure Lengend Snippet: Fig. 4. NS1 partially blocks the binding of p53 at the level of p53-responsive genes, in a promoter-dependent manner. H1299 cells were co-transfected with 4 lg of pSV-p53 and 4 lg of pCI-empty or pCI-NS1-H3N2 expression plasmid. After 24 h, cells were fixed with 1% formaldehyde for 10 min at room temperature. The cells were scraped and washed with ice-cold PBS, and then sonicated with salmon sperm DNA and protease inhibitors. Immunoprecipitation was carried out using Dynabeads (Invitrogen) and different antibodies produced in mouse: an anti-p53 monoclonal antibody (DO-1), or an anti-NS1 monoclonal antibody (Santa-Cruz ref sc-130568). Mouse IgGs were used as negative controls. The amount of total input DNA per chip was adjusted to 25 lg. After immunoprecipitation and DNA purification, samples were analysed by RT-qPCR with specific primers and probes targeting p21 and Bax. Mean values ± standard deviation from two independent experiments are presented. (ns, P > 0.05; ⁄⁄P < 0.005).

Article Snippet: A mouse anti-NS1 monoclonal antibody (Santa-Cruz ref sc-130568) was used to control a possible binding of NS1 to promoter regions.

Techniques: Binding Assay, Transfection, Expressing, Plasmid Preparation, Sonication, Immunoprecipitation, Produced, Quantitative RT-PCR, Standard Deviation

Journal: iScience

Article Title: Fine-tuned KDM1A alternative splicing regulates human cardiomyogenesis through an enzymatic-independent mechanism

doi: 10.1016/j.isci.2022.104665

Figure Lengend Snippet: Biochemical and enzymatic properties of KDM1A isoforms (A) Schematic of KDM1A and CoREST constructs. (B) Crystal structure of a KDM1A/CoREST (PDB: 2v1d ). (C) Close up view of Pro171, the first ordered residue in the full length KDM1A of the KDM1A+2a/CoREST complex. The arrow outlines the site of E2a insertion after Gly173. The weighted 2Fo-Fc electron density is contoured at 1 σ level. The backbone electron density is interrupted just at the beginning of the region subjected to alternative splicing. (D) Chromatograms showing the macromolecular species detected after the incubation of KDM1A/CoREST with the nucleosomes containing a propargyl group at the position 4 of H3. (E) The affinity of KDM1A/CoREST (blue) and KDM1A+2a (red) for a peptide comprising nine N-terminal residues of SNAI1 is evaluated through fluorescence polarization assay. (F) Venn diagram of the commonly enriched peptides identified by mass spectrometry (MS) in the FLAG-immunoprecipitates. (G) MS peptide counts identified in KDM1A and IgG co-immunoprecipitates from total lysates of KDM1A −/− hESCs in FLAG-ubKDM1A (FLAG-ub R ) or FLAG-KDM1A+2a (FLAG-E2a R ). (H) Western blot analysis of IgG, HDAC1, HDAC2, T7-ubKDM1A and T7-KDM1A+2a from hESC lysates. See also Tables S1–S3 .

Article Snippet: Rabbit anti-HDAC1 , Abcam , Cat#ab19845; RRID: AB_470299.

Techniques: Construct, Incubation, Fluorescence, Mass Spectrometry, Western Blot

Journal: iScience

Article Title: Fine-tuned KDM1A alternative splicing regulates human cardiomyogenesis through an enzymatic-independent mechanism

doi: 10.1016/j.isci.2022.104665

Figure Lengend Snippet:

Article Snippet: Rabbit anti-HDAC1 , Abcam , Cat#ab19845; RRID: AB_470299.

Techniques: Recombinant, Transfection, Protease Inhibitor, Magnetic Beads, Staining, Immunocytochemistry, Calcium Assay, Purification, Western Blot, Plasmid Preparation, Software, Electrophoresis, Microscopy, Imaging, Real-time Polymerase Chain Reaction, Mass Spectrometry

Journal: bioRxiv

Article Title: Direct effects on endothelial cells are essential for perivascular cell (pericyte)-dependent amplification of orthoflavivirus NS1-mediated microvascular leakage

doi: 10.64898/2026.01.29.702573

Figure Lengend Snippet: (A) Representative images of HUVECs forming vessel-like structures in 3D Geltrex® matrix in the presence of HSCs, with or without 1 µg/mL NS1 treatment. Scale bars, 100 µm. (B) Angiogenesis assay quantification method. “Junctions” connect vessel-like structures; “branches” connect to junctions at one end; “segments” connect at both ends. “Total branching length” includes all branches; counts reflect the total number of segments, branches and junctions within field of view. (C-F) Quantification of vessel-like structures in pericyte-endothelial cell cocultures following NS1 treatment, normalised to untreated HUVECs (dashed line). Data points represent technical replicates within an experiment with different symbol shapes corresponding to different experiments (N=4, n=4). Bars represent the mean, and error bars represent SEM. Indicated significance is for treatment versus untreated cocultures (Untreated). * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Plasmids encoding Streptavidin-tagged NS1 from AHFV (pKM74; viral reference sequence NCBI accession JF416957.1 ) or KFDV (pKM79 accession JF416958.1 ) were transfected into CHO DG44 cells using FreeStyle MAX reagent (ThermoFisher), followed by selection using 0.3 μg/mL geneticin (G418 sulphate, ThermoFisher).

Techniques: Angiogenesis Assay

Journal: bioRxiv

Article Title: Direct effects on endothelial cells are essential for perivascular cell (pericyte)-dependent amplification of orthoflavivirus NS1-mediated microvascular leakage

doi: 10.64898/2026.01.29.702573

Figure Lengend Snippet: (A-B) ECIS measurement of permeability of monocultures of HUVECs (A) or LSECs (B) treated with preconditioned media from HSCs treated with 1 µg/mL NS1 or 100 ng/mL TNF-α for 24 h; “Untreated” refers to HUVECs or LSECs treated with HSC media only and the “EC only” refers to untreated HUVECs or LSECs without the addition of HSC media. (C, D) Dextran permeability assay measuring HUVEC (C) or LSEC (D) permeability in coculture with HSCs 24 h post-treatment with 1 µg/mL NS1, 100 ng/mL TNF-α (positive control), or eluate (negative) control. Data points represent technical replicates within an experiment with different symbol shapes corresponding to different experiments (N=3, n=3). Bars represent the mean, and error bars represent SEM. (E, F) TEER measurement of semi-contact cocultures of HUVECs (A) or LSECs (B) cultured with HSCs and treated with 1 µg/mL NS1, 100 ng/mL TNF-α (positive control), or eluate (negative) control. Permeability measurements were normalised to each sample pre-treatment for ECIS data (A, B) or the untreated endothelial cell only control (EC only; dashed line) for EVOM data (E, F). In the time course experiments (A, B, E, F), each data point represents the mean ± SEM. In all experiments indicated significance is compared to untreated cocultures; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Plasmids encoding Streptavidin-tagged NS1 from AHFV (pKM74; viral reference sequence NCBI accession JF416957.1 ) or KFDV (pKM79 accession JF416958.1 ) were transfected into CHO DG44 cells using FreeStyle MAX reagent (ThermoFisher), followed by selection using 0.3 μg/mL geneticin (G418 sulphate, ThermoFisher).

Techniques: Permeability, FITC-Dextran Permeability Assay, Positive Control, Negative Control, Cell Culture, Control

Journal: bioRxiv

Article Title: Direct effects on endothelial cells are essential for perivascular cell (pericyte)-dependent amplification of orthoflavivirus NS1-mediated microvascular leakage

doi: 10.64898/2026.01.29.702573

Figure Lengend Snippet: Endothelial barrier integrity measurements for HUVEC (A, B) or LSEC (C) monocultures following treatment with 1 µg/mL wild-type DENV-2, AHFV or KFDV NS1, or the nonfunctional DENV-2 NS207Q mutant (negative control), or 100 ng/mL TNF-α (positive control), or eluate (negative) control as indicated. Permeability measurements were normalised to the untreated endothelial cell only control (Untreated) for EVOM TEER data (A), or to each sample before treatment for ECIS data (B, C). Each data point represents the mean ± SEM. Indicated significance is for treatment versus untreated EC only control (Untreated). * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Plasmids encoding Streptavidin-tagged NS1 from AHFV (pKM74; viral reference sequence NCBI accession JF416957.1 ) or KFDV (pKM79 accession JF416958.1 ) were transfected into CHO DG44 cells using FreeStyle MAX reagent (ThermoFisher), followed by selection using 0.3 μg/mL geneticin (G418 sulphate, ThermoFisher).

Techniques: Mutagenesis, Negative Control, Positive Control, Permeability, Control

Journal: Frontiers in Microbiology

Article Title: NS Segment of a 1918 Influenza A Virus-Descendent Enhances Replication of H1N1pdm09 and Virus-Induced Cellular Immune Response in Mammalian and Avian Systems

doi: 10.3389/fmicb.2018.00526

Figure Lengend Snippet: Viral expression of different NS1 proteins and their ability to regulate IFN-β induction. (A) NS1 expression 24 h p.i. in A549 infected with Gi-wt, Gi-NS-PR8, and Gi-NS-Vict (MOI of 1) was detected by Western blot. (B) Effect on IFN-β promoter-induction by transiently expressed NS1 of Gi-wt (NS-Gi), PR8 (NS-PR8) and Victoria (NS-Vict) was analyzed. 293T cells were transfected with pMP-NS-Gi, pMP-NS-PR8, pMP-NS-Vict or empty pHW2000 (Mock and control) together with a Firefly luciferase expression plasmid under the control of IFN-β promoter (p125-Luc) and Renilla luciferase expression plasmid (pRL-SV40). Except for the mock, the transfected cells were either infected with NDV (MOI = 3) or treated with 50 ng/ml recombinant human TNF-α for the induction of IFN-β promoter. After 24 h p.i. the cells were lysed and assayed for luciferase activity. Statistical analysis was performed using “student t -test” test. ∗ p < 0.05, ∗∗ p < 0.001, ∗∗∗ p < 0.0001; ns, non-significant. Error bars represent standard deviation (SD).

Article Snippet: Afterward, detection of the viral NS proteins was achieved using mouse monoclonal antibodies recognizing influenza A virus NS1 (Abcam, Cambridge, United Kingdom) diluted 1:2000 in blocking buffer.

Techniques: Expressing, Infection, Western Blot, Transfection, Luciferase, Plasmid Preparation, Recombinant, Activity Assay, Standard Deviation

Journal: Cell reports

Article Title: Flavivirus NS1 Triggers Tissue-Specific Vascular Endothelial Dysfunction Reflecting Disease Tropism

doi: 10.1016/j.celrep.2019.01.036

Figure Lengend Snippet: (A-E) Human endothelial cells from different tissues (A, lung; B, skin; C, umbilical vein; D, brain; and E, liver) were grown on Transwell semi-permeable membranes (0.4 μm pore size), and distinct flavivirus NS1 proteins (5 μg/mL, 1.5 μg total protein) were added to the apical chamber. TNF-α (10 ng/mL) was used as a positive control, and DENV2 recombinant envelope (E) protein (5 μg/mL) was used as a negative control. A TEER assay was used to evaluate the effect of these NS1 proteins on endothelial permeability at indicated time-points over 48 h. ^ represents media change. Relative TEER values from two independent experiments performed in duplicate are plotted. Error bars indicate SEM. Statistically significant differences between distinct groups (all time points combined for each group) compared to the untreated groups were determined by a two-way ANOVA analysis using Dunnett’s test for multiple comparisons, with ****p < 0.0001 and ns (not significant), p > 0.05.

Article Snippet: Recombinant West Nile virus NS1 (NY99) protein produced in 293 human cells. C-terminal 6x His-Tag , The Native Antigen Company , NA.

Techniques: Pore Size, Positive Control, Recombinant, Negative Control, Permeability

Journal: Cell reports

Article Title: Flavivirus NS1 Triggers Tissue-Specific Vascular Endothelial Dysfunction Reflecting Disease Tropism

doi: 10.1016/j.celrep.2019.01.036

Figure Lengend Snippet: Binding of flavivirus NS1 proteins to the surface of different human endothelial cell monolayers as measured by (A-E) confocal microscopy or (F-J) western blot. (A-E) (A) HPMECs, (B) HMEC-1, (C) HUVECs, (D) HBMECs, and (E) HLSECs were grown on coverslips and treated with 5 μg/mL NS1 or10 μg/mL NS1 (1.5 μg or 3 μg of total protein, respectively) as indicated (also see ); binding was evaluated 1 h post-treatment (hpt). The amount of bound NS1 was quantified and expressed as MFI. (F-J) Cell lysates from (F) HPMECs, (G) HMEC-1, (H) HUVECs, (I) HBMECs, and (J) HLSECs treated with 10 μg/mL (3 μg total protein) of flavivirus NS1 protein and collected 1 hpt and analyzed by western blot . All graphs show the average of quantification from two independent experiments run in duplicate. Error bars indicate SEM. In (A)-(E), statistically significant differences between distinct treatment groups compared to the untreated group [NS1(−)] were determined by a two-way ANOVA analysis using Dunnett’s test for multiple comparisons, with *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: Recombinant West Nile virus NS1 (NY99) protein produced in 293 human cells. C-terminal 6x His-Tag , The Native Antigen Company , NA.

Techniques: Binding Assay, Confocal Microscopy, Western Blot

Journal: Cell reports

Article Title: Flavivirus NS1 Triggers Tissue-Specific Vascular Endothelial Dysfunction Reflecting Disease Tropism

doi: 10.1016/j.celrep.2019.01.036

Figure Lengend Snippet: (A) Sialic acid expression on human endothelial cell monolayers grown on coverslips 6 hpt with different flavivirus NS1 proteins (5 μg/mL, 1–5 μg total protein), examined by confocal microscopy. Sialic acid was stained with wheat germ agglutinin conjugated to Alexa Fluor 647 (red). Nuclei were stained with Hoechst (blue). Images (20×) are representative of three independent experiments. Scale bar, 10 μm. (B) Quantification of MFI in (A) from three independent experiments. Error bars indicate SEM. Reduction of sialic acid expression in NS1-treated monolayers was normalized to untreated controls (dotted line). Statistically significant differences between distinct treatment groups compared to the untreated group were determined by a two-way ANOVA analysis using Dunnett’s test for multiple comparisons, with *p < 0.05, ***p < 0.001, and ****p < 0.0001.

Article Snippet: Recombinant West Nile virus NS1 (NY99) protein produced in 293 human cells. C-terminal 6x His-Tag , The Native Antigen Company , NA.

Techniques: Expressing, Confocal Microscopy, Staining

Journal: Cell reports

Article Title: Flavivirus NS1 Triggers Tissue-Specific Vascular Endothelial Dysfunction Reflecting Disease Tropism

doi: 10.1016/j.celrep.2019.01.036

Figure Lengend Snippet: (A) Heparan sulfate expression on human endothelial cell monolayers grown on coverslips 6 hpt with different flavivirus NS1 proteins (5 μg/mL, 1–5 μg total protein), examined by confocal microscopy. Heparan sulfate was stained with anti-heparan sulfate mAb (green). Nuclei were stained with Hoechst (blue). Images (20×) are representative of three independent experiments. Scale bar, 10 μm. (B) Quantification of MFI in (A) from three independent experiments. Error bars indicate SEM. Reduction of heparan sulfate expression in NS1-treated monolayers was normalized against untreated controls (dotted line). Statistically significant differences between distinct treatment groups compared to the untreated group were determined by a two-way ANOVA analysis using Dunnett’s test for multiple comparisons, with *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: Recombinant West Nile virus NS1 (NY99) protein produced in 293 human cells. C-terminal 6x His-Tag , The Native Antigen Company , NA.

Techniques: Expressing, Confocal Microscopy, Staining

Journal: Cell reports

Article Title: Flavivirus NS1 Triggers Tissue-Specific Vascular Endothelial Dysfunction Reflecting Disease Tropism

doi: 10.1016/j.celrep.2019.01.036

Figure Lengend Snippet: (A and B) Fold change over untreated controls (dotted lines) of the quantification of MFI from three independent confocal microscopy experiments examining (A) expression and (B) activity of cathepsin L (Magic Red reagent) in human endothelial cell monolayers 6 hpt with different flavivirus NS1 proteins (5 μg/mL, 1–5 μg total protein) . (C) Western blot data of pro- and mature- cathepsin L expression in human endothelial cell monolayers 6 hpt with different flavivirus NS1 proteins (5 μg/mL, 1.5 μg total protein). (D and E) Densitometry of bands from (C) for (D) pro-cathepsin L and (E) mature cathepsin L with values normalized to GAPDH and expressed as fold change from untreated monolayers. (F) Fold change over untreated controls (dotted line) of the quantification of MFI from three independent confocal microscopy experiments examining the expression of heparanase in human endothelial cell monolayers 6 hpt with different flavivirus NS1 proteins (5 μg/mL, 1.5 μg total protein) . For all graphs, error bars indicate SEM. Statistically significant differences between distinct treatment groups compared to the untreated group were determined by a two-way ANOVA analysis using Dunnett’s test for multiple comparisons, with *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: Recombinant West Nile virus NS1 (NY99) protein produced in 293 human cells. C-terminal 6x His-Tag , The Native Antigen Company , NA.

Techniques: Confocal Microscopy, Expressing, Activity Assay, Western Blot

Journal: Cell reports

Article Title: Flavivirus NS1 Triggers Tissue-Specific Vascular Endothelial Dysfunction Reflecting Disease Tropism

doi: 10.1016/j.celrep.2019.01.036

Figure Lengend Snippet: (A-H) Wild-type C57BL/6 mice were used to evaluate (A and B) local or (C-H) systemic vascular leakage induced by flavivirus NS1 proteins injected either (A and B) intradermally (dorsal dermis) or (C-H) intravenously with 15 μg and 200 μg (10 mg/kg) of total protein, respectively. The magnitude of the vascular leakage induced by these proteins was evaluated by using (A, B, and E-H) Alexa-Fluor-680-conjugated dextran (10 mg/mL) or (C and D) Evans blue dye (0.5%) and quantified by (A, B, and E-H) fluorescent imaging or (C and D) spectrophotometric analyses. (A, E, and F) Images (LI-COR Odyssey) and (B-D, G, and H) MFI values are representative of two individual experiments (n = 3–6 per group). Ovalbumin (OVA; 10 mg/kg) and PBS were used as controls for systemic and local vascular leakage experiments, respectively. (I) Levels of heparan sulfate detected in serum of mice (n = 3 per group) treated with ovalbumin (OVA), DENV2 NS1, WNV NS1, or YFV NS1 (all 10 mg/kg) as measured by ELISA. Untreated mice were used as a steady-state control. For dextran-adapted dermal Miles assay, data were derived from three independent experiments and expressed as the fold-change ratio of the MFI obtained in each individual treatment group compared to PBS injection (dotted line in B). MFI was measured inside the leakage area by drawing a standard circular area in each treatment condition using ImageJ tools. Statistically significant differences between distinct treatment groups compared to the control groups were determined by a one-way ANOVA analysis using Dunnett’s test for multiple comparisons, with *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and ns (not significant), p > 0.05 . Error bars indicate SEM. Scale bars represent (A) 2.5 cm and (E and F) 0.5 cm.

Article Snippet: Recombinant West Nile virus NS1 (NY99) protein produced in 293 human cells. C-terminal 6x His-Tag , The Native Antigen Company , NA.

Techniques: Injection, Imaging, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: Cell reports

Article Title: Flavivirus NS1 Triggers Tissue-Specific Vascular Endothelial Dysfunction Reflecting Disease Tropism

doi: 10.1016/j.celrep.2019.01.036

Figure Lengend Snippet: Flavivirus infections can affect specific tissues, including brain, lung, skin, liver, and the placenta during pregnancy, to cause neurotropic-encephalitic or systemic diseases in humans. Infection with any of the flaviviruses leads to secretion of NS1, a soluble viral protein that directly triggers endothelial hyperpermeability and vascular leakage associated with the disruption of key components of the endothelial glycocalyx layer, which lines the luminal surface of endothelial cells in the vasculature. The EGL is composed of monosaccharides such as sialic acid (Sia), glycosaminoglycans (GAGs) such as heparan sulfate, and heparan sulfate proteoglycans (HSPGs) such as syndecan-1. Here, we describe an endothelial cell-type-dependent increase in permeability and vascular leakage induced by different flavivirus NS1 proteins that reflect the disease pathogenesis of distinct flavivirus infections. DENV NS1 increases the permeability of endothelial cells from multiple tissues, such as lung, skin, umbilical vein, brain, and liver, consistent with the systemic disease caused by DENV. ZIKV NS1 induces endothelial dysfunction of umbilical vein and brain endothelial cells, reflecting severe congenital and neurological defects associated with Zika, while WNV and JEV NS1 only affect the barrier function of brain endothelial cells, in line with the neurotropic and encephalitic nature of WNV and JEV disease. Finally, YFV NS1 increases the permeability of lung and especially liver endothelial cells, consistent with hepatic and systemic pathology of yellow fever virus. This selective permeability may contribute to the pathogenesis of the different flaviviruses by either inducing extravasation of fluids that result in inflammation of tissues or facilitating virus dissemination into target organs that may lead to enhanced viral infection and disease.

Article Snippet: Recombinant West Nile virus NS1 (NY99) protein produced in 293 human cells. C-terminal 6x His-Tag , The Native Antigen Company , NA.

Techniques: Infection, Disruption, Permeability, Virus

Journal: Cell reports

Article Title: Flavivirus NS1 Triggers Tissue-Specific Vascular Endothelial Dysfunction Reflecting Disease Tropism

doi: 10.1016/j.celrep.2019.01.036

Figure Lengend Snippet:

Article Snippet: Recombinant West Nile virus NS1 (NY99) protein produced in 293 human cells. C-terminal 6x His-Tag , The Native Antigen Company , NA.

Techniques: Purification, Marker, Recombinant, Virus, Produced, Membrane, Antibody Labeling, Cell Culture, Plasmid Preparation, Software