Journal: Journal of Virology

Article Title: Glycosylated NS3/NS3A protein of bluetongue virus facilitates efficient viral egress via lipid raft anchoring

doi: 10.1128/jvi.02144-25

Figure Lengend Snippet: N-linked glycosylation drives plasma membrane accumulation of NS3/NS3A. ( A ) The stability of NS3/NS3A WT and NS3/NS3A N150Q proteins was examined in both transfected (top panels) and BTV-20-infected cells (bottom panels; MOI = 10). For transfection assays, HEK-293T cells were transfected with plasmids expressing NS3/NS3A WT or the N150Q mutant and treated with cycloheximide (CHX; 100 μg/mL) at 18 h post-transfection (designated as 0 h post-CHX treatment) to block de novo protein synthesis. For infection assays, MDOK cells were infected with BTV-20 WT or BTV-20 N150Q and treated with CHX (100 μg/mL) at 10 h post-infection (designated as 0 h post-CHX treatment). Cells were harvested at the indicated time points and analyzed by Western blotting. ( B ) Quantification of NS3/NS3A protein levels shown in panel A was performed by ImageJ densitometric analysis. Protein levels at each time point were normalized to the corresponding 0 h post-CHX treatment (18 h post-transfection or 10 h post-infection, respectively). ( C ) Subcellular localization of NS3/NS3A in MDOK cells infected with BTV-20 WT or BTV-20 N150Q (MOI = 5, 12 h.p.i.). NS3/NS3A (red) was co-stained with ER marker anti-calnexin (green) or Golgi marker anti-syntaxin 6 (green). Fluorescence distribution was evaluated using line-scan intensity profiles. Scale bar, 5 µm. ( D ) Subcellular localization of NS3/NS3A in MDOK cells infected with BTV-20 WT or BTV-20 N150Q (MOI = 5, 12 h.p.i.). NS3/NS3A (red) was co-stained with plasma membrane marker WGA-Alexa Fluor 488 (green). Line-scan intensity profiles are shown. Scale bar, 5 µm. ( E ) Plasma membrane isolation of HEK-293T cells transfected with NS3/NS3A WT or N150Q mutant, followed by Western blot analysis. PM (plasma membrane fraction); NPM (non-plasma membrane fraction); Total (plasma membrane fraction + non-plasma membrane fraction). ( F ) Quantification of NS3/NS3A at the plasma membrane fraction was analyzed by Image J from panel E (* P < 0.05, two-tailed unpaired t-test). ( G ) Confocal imaging of HeLa cells co-transfected with NS3/NS3A (WT or N150Q, red) and VP2 (green) or VP5 (green), showing their subcellular colocalization. Colocalization was assessed by line-scan intensity profiles.

Article Snippet: Commercial antibodies used in this study included anti-FLAG (DYKDDDDK) monoclonal antibody (1:1,000 for immunofluorescence assay [IFA], 1:10,000 for western blotting [WB]; 66008-4-Ig, Proteintech), anti-HA polyclonal antibody (1:100 for IFA, 1:1,000 for WB; 51064-2-AP, Proteintech), anti-β-actin monoclonal antibody (1:10,000 for WB; 66009-1-Ig, Proteintech), anti-Calnexin polyclonal antibody (1:200 for IFA; 10427-2-AP, Proteintech), anti-Syntaxin 6 polyclonal antibody (1:200 for IFA; 10841-1-AP, Proteintech), anti-Flotillin 1 monoclonal antibody (1:100 for IFA; 67968-1-Ig, Proteintech), and anti-Filamin A (FLNA) monoclonal antibody (1:1,000 for WB; 67133-1-Ig, Proteintech).

Techniques: Glycoproteomics, Clinical Proteomics, Membrane, Transfection, Infection, Expressing, Mutagenesis, Blocking Assay, Western Blot, Staining, Marker, Fluorescence, Isolation, Two Tailed Test, Imaging

Journal: Journal of Virology

Article Title: Glycosylated NS3/NS3A protein of bluetongue virus facilitates efficient viral egress via lipid raft anchoring

doi: 10.1128/jvi.02144-25

Figure Lengend Snippet: N-linked glycosylation of NS3/NS3A facilitates its raft-enriched membrane association and efficient BTV release. ( A ) Representative confocal images of HeLa cells transfected with NS3/NS3A WT or the N150Q mutant. Cells were fixed and stained 20 h post-transfection. NS3/NS3A was detected using anti-NS3/NS3A (red), and lipid raft-enriched membrane domains were labeled with anti-flotillin-1 (green). Scale bars, 5 μm. ( B and C ) Confocal images of HeLa cells expressing NS3/NS3A treated with trifluoperazine (TFP) ( B ) or methyl-β-cyclodextrin (MβCD) ( C ). Cells were fixed and stained 20 h post-transfection. NS3/NS3A is shown in red and flotillin-1 in green. Fluorescence intensity profiles were analyzed along the indicated line scans. Scale bars, 5 μm. ( D ) Localization of NS3/NS3A at the plasma membrane of HeLa cells with or without MβCD or TFP treatment. The plasma membrane was stained using WGA-Alexa Fluor 488 (green), and NS3/NS3A was visualized in red. Scale bars, 5 μm. ( E ) Quantification of plasma membrane (PM) localization from panel D, shown as the ratio of PM-associated NS3/NS3A fluorescence intensity (defined by WGA staining) to total cellular NS3/NS3A intensity. Values were normalized to vehicle-treated controls and are presented as mean ± SD. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test (** P < 0.01, **** P < 0.0001). ( F ) MDOK or HEK-293T cells were infected with BTV-20 WT or BTV-20 N150Q at an MOI of 5. At 6 h post-infection, the cells were treated with MβCD or TFP, and viral titers were determined at 12 h post-infection. Data represent mean ± SD ( n = 3 biological replicates). Statistical significance was determined by two-way ANOVA with Šídák’s multiple comparisons test (ns, not significant; *** P < 0.001; **** P < 0.0001). ( G ) Analysis of BTV-20 release efficiency following raft disruption. MDOK cells were infected with BTV-20 (MOI = 5) and then treated with MβCD or TFP. Extracellular and intracellular viral titers were determined at the indicated time points. Release efficiency was calculated as [extracellular titer/(extracellular + intracellular titer)] × 100%. Data are presented as mean ± SD ( n = 3 biological replicates). Statistical significance was determined using two-way ANOVA with Šídák’s multiple comparisons test (ns, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). ( H ) A total of 1,136 host proteins were identified by quantitative proteomics, of which 102 proteins were enriched in the NS3/NS3A WT interactome relative to the N150Q mutant (log₂ FC > 4 or unique to WT). Highlighted are the top eight candidates. ( I ) Gene Ontology (GO) enrichment analysis of proteins enriched in the NS3/NS3A WT interactome. Significantly overrepresented terms are shown for the biological process, cellular component, and molecular function categories. Bars represent −log₁₀ ( P value). ( J ) Sucrose gradient fractionation followed by immunoblotting of lysates from transfected HEK-293T cells, or from infected MDOK cells. DRM (raft-enriched) fractions correspond to fractions 5–12, whereas detergent-soluble membrane (DSM) fractions correspond to fractions 13–20. ( K ) Co-immunoprecipitation analysis of NS3/NS3A WT , NS3/NS3A N150Q , and FLNA. Cells were transfected with NS3/NS3A variants, followed by Co-IP.

Article Snippet: Commercial antibodies used in this study included anti-FLAG (DYKDDDDK) monoclonal antibody (1:1,000 for immunofluorescence assay [IFA], 1:10,000 for western blotting [WB]; 66008-4-Ig, Proteintech), anti-HA polyclonal antibody (1:100 for IFA, 1:1,000 for WB; 51064-2-AP, Proteintech), anti-β-actin monoclonal antibody (1:10,000 for WB; 66009-1-Ig, Proteintech), anti-Calnexin polyclonal antibody (1:200 for IFA; 10427-2-AP, Proteintech), anti-Syntaxin 6 polyclonal antibody (1:200 for IFA; 10841-1-AP, Proteintech), anti-Flotillin 1 monoclonal antibody (1:100 for IFA; 67968-1-Ig, Proteintech), and anti-Filamin A (FLNA) monoclonal antibody (1:1,000 for WB; 67133-1-Ig, Proteintech).

Techniques: Glycoproteomics, Membrane, Transfection, Mutagenesis, Staining, Labeling, Expressing, Fluorescence, Clinical Proteomics, Infection, Disruption, Quantitative Proteomics, Fractionation, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Aging Cell

Article Title: miR ‐375‐3p/ STX6 Exacerbates Atherosclerosis by Promoting Endothelial Cell Senescence via Activation of TGF ‐Beta Signals

doi: 10.1111/acel.70326

Figure Lengend Snippet: STX6 is a target of miR‐375‐3p in endothelial cell senescence. (a) Predicted and mutated miR‐375‐3p binding sites within the 3′UTR of STX6. (b) Luciferase assay of WT and mutant STX6 3′UTR reporters in HEK293A cells transfected with miR‐375‐3p mimic or scramble. (c) qRT‐PCR analysis of STX6 mRNA in HUVECs transfected with miR‐375‐3p mimic or scramble, normalized to GAPDH. (d, e) Western blot analysis of STX6 protein expression in HUVECs transfected with miR‐375‐3p mimic (d) or STX6 siRNAs (e), normalized to GAPDH. (f) Representative SA‐β‐gal staining and quantification in HUVECs transfected with STX6 siRNAs or scramble. Scale bar = 200 μm. (g) Representative colony formation images showing cell proliferation in HUVECs transfected with STX6 siRNAs or scramble. Scale bar = 5 mm. (h) qRT‐PCR analysis of p15, IL6, and IL8 mRNA in HUVECs transfected with STX6 siRNAs or scramble, normalized to GAPDH. Data are presented as mean ± SD; statistical significance determined by unpaired two‐tailed Student's t ‐test. ** p < 0.01, *** p < 0.001.

Article Snippet: The primary antibodies included STX6, GAPDH (Proteintech, Wuhan, Hubei, China), p‐SMAD2, SMAD2 (Cell Signaling Technology, Boston, MA, USA), p15 (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Binding Assay, Luciferase, Mutagenesis, Transfection, Quantitative RT-PCR, Western Blot, Expressing, Staining, Two Tailed Test

Journal: Aging Cell

Article Title: miR ‐375‐3p/ STX6 Exacerbates Atherosclerosis by Promoting Endothelial Cell Senescence via Activation of TGF ‐Beta Signals

doi: 10.1111/acel.70326

Figure Lengend Snippet: STX6 overexpression rescues the pro‐senescent effect of miR‐375‐3p. (a) Representative SA‐β‐gal staining and quantification in HUVECs infected with Ad‐STX6 and transfected with miR‐375‐3p mimic. Scale bar = 200 μm. (b) qRT‐PCR analysis of p15, IL6, and IL8 mRNA expression in HUVECs infected with Ad‐STX6 and transfected with miR‐375‐3p mimic, normalized to GAPDH. (c) Colony formation assay showing cell proliferation in HUVECs infected with Ad‐STX6 and transfected with miR‐375‐3p mimic. Scale bar = 5 mm. Data are presented as mean ± SD; statistical significance determined by two‐way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The primary antibodies included STX6, GAPDH (Proteintech, Wuhan, Hubei, China), p‐SMAD2, SMAD2 (Cell Signaling Technology, Boston, MA, USA), p15 (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Over Expression, Staining, Infection, Transfection, Quantitative RT-PCR, Expressing, Colony Assay

Journal: Aging Cell

Article Title: miR ‐375‐3p/ STX6 Exacerbates Atherosclerosis by Promoting Endothelial Cell Senescence via Activation of TGF ‐Beta Signals

doi: 10.1111/acel.70326

Figure Lengend Snippet: miR‐375‐3p/STX6 signaling promotes endothelial cell senescence through the increased internalization of TGFBR1. (a) Immunofluorescence images showing colocalization (yellow) of TGFBR1 (green) and EEA1 (red) in HUVECs infected with Ad‐STX6 and transfected with miR‐375‐3p. Nuclei stained with Hoechst (blue). Scale bar = 5 μm. (b–d) Western blot analysis of SMAD2 phosphorylation and p15 expression in HUVECs transfected with miR‐375‐3p mimic or STX6 siRNAs (b), infected with Ad‐STX6 and transfected with miR‐375‐3p mimic (c), or co‐transfected with SMAD2 siRNA and miR‐375‐3p mimic or STX6 siRNAs (d). (e) Representative SA‐β‐gal staining and quantification in HUVECs co‐transfected with SMAD2 siRNA and miR‐375‐3p mimic or STX6 siRNAs. Scale bar = 200 μm. (f, g) Western blot (f) and SA‐β‐gal (g) staining in HUVECs infected with Ad‐STX6 and stimulated with TGF‐β1. Scale bar = 200 μm. Data are presented as mean ± SD; statistical significance determined by unpaired two‐tailed Student's t ‐test or two‐way ANOVA. ** p < 0.01, *** p < 0.001.

Article Snippet: The primary antibodies included STX6, GAPDH (Proteintech, Wuhan, Hubei, China), p‐SMAD2, SMAD2 (Cell Signaling Technology, Boston, MA, USA), p15 (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Immunofluorescence, Infection, Transfection, Staining, Western Blot, Phospho-proteomics, Expressing, Two Tailed Test

Journal: Aging Cell

Article Title: miR ‐375‐3p/ STX6 Exacerbates Atherosclerosis by Promoting Endothelial Cell Senescence via Activation of TGF ‐Beta Signals

doi: 10.1111/acel.70326

Figure Lengend Snippet: Overexpression of STX6 reduce atherosclerotic plaque in mice. (a) qRT‐PCR analysis of Stx6 expression in aortas of WT ( n = 21) and ApoE −/− ( n = 23) mice fed HFD for 8 weeks, normalized to Gapdh. (b) Schematic of experimental design showing Ad‐Ctrl or Ad‐STX6 injection (1 × 10 9 PFU per mouse, twice weekly for 8 weeks) in ApoE −/− mice on HFD. (c) Representative Oil Red O staining images and quantification of lesion size and positive area in the aortic sinus. Scale bar = 200 μm. (d) Representative Oil Red O staining images and quantification of plaque area in the entire aortic tree. (e) Immunofluorescence staining of macrophage marker MOMA2 (green) in aortic sinus. Scale bar = 200 μm. (f) Representative SA‐β‐gal and Cd31 co‐staining in aortic sinus lesions. Scale bar = 5 μm. (g) Immunofluorescence of phosphorylated Smad2 (green) and Cd31 (red) in the aortic intima (L). Nuclei stained with Hoechst (blue). Scale bar = 5 μm. Data are presented as mean ± SD; statistical significance determined by unpaired two‐tailed Student's t ‐test. * p < 0.05, ** p < 0.01.

Article Snippet: The primary antibodies included STX6, GAPDH (Proteintech, Wuhan, Hubei, China), p‐SMAD2, SMAD2 (Cell Signaling Technology, Boston, MA, USA), p15 (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Over Expression, Quantitative RT-PCR, Expressing, Injection, Staining, Immunofluorescence, Marker, Two Tailed Test