293t  (ATCC)

Journal: Bioactive Materials

Article Title: Cord blood natural killer cell-derived extracellular vesicles inhibit Zika virus infectivity through ITGB2/perforin-mediated envelope disruption in vitro and in vivo

doi: 10.1016/j.bioactmat.2026.01.030

Figure Lengend Snippet: Perforin mediates the direct virion-disrupting activity of CBNK-EVs. (a) Quantification of perforin and granzyme B in CBNK-EVs by ELISA. (b) Vero-E6 cells were infected with ZIKV (MOI = 1) after pre-incubation of viral particles with 40 ng/L of recombinant perforin or granzyme B for 2 h. ZIKV E protein levels were assessed by Western blot after 24 h, densitometric analysis of the protein bands is shown (right, n = 3). (c) CBNK-EVs were pre-incubated with ZIKV in the presence or absence of 10 mM EGTA, followed by micrococcal nuclease digestion. Protected ZIKV E RNA was quantified by RT-qPCR to assess virion integrity. (d) Western blot analysis of perforin expression in CBNK cells after transfection with the indicated siRNAs. (e) Quantification of perforin levels from (d). (f) Perforin levels in CBNK-EVs collected from control or perforin-knockdown cells, measured by ELISA and normalized to particle count (per 10 11 particles, n = 6). (g) Vero-E6 cells were infected with ZIKV (MOI = 1) that had been pre-incubated with control or perforin-knockdown CBNK-EVs. ZIKV E protein levels were evaluated by Western blot. (h) Analysis of ZIKV E protein levels from (g). (i) Characterization of ITGB2-EVs and control EVs from 293T cells by NTA and TEM. Scale bar, 100 nm. (j) Zeta potential measurements of ITGB2-EVs and control EVs. (k) Western blot analysis of EV markers and ITGB2 expression in ITGB2-EVs and control EVs. (l) Antiviral activity of CBNK-EVs and ITGB2-EVs evaluated by cell-based ZIKV E protein ELISA. Data are presented as mean ± SD (n = 3). ∗P < 0.05, ∗∗P < 0.01, ∗P < 0.001 (one-way ANOVA).

Article Snippet: Vero E6, BHK21, 293T and HTR-8/Svneo cells were purchased from ATCC.

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Infection, Incubation, Recombinant, Western Blot, Quantitative RT-PCR, Expressing, Transfection, Control, Knockdown, Zeta Potential Analyzer

Journal: Bioactive Materials

Article Title: Cord blood natural killer cell-derived extracellular vesicles inhibit Zika virus infectivity through ITGB2/perforin-mediated envelope disruption in vitro and in vivo

doi: 10.1016/j.bioactmat.2026.01.030

Figure Lengend Snippet: ITGB2 facilitates CBNK-EVs binding to Zika virions and enhances cellular susceptibility to ZIKV. (a) Multicolor immunofluorescence staining of 293T cells transfected with ITGB2, showing co-localization (yellow) between ITGB2 (green) and ZIKV E protein (red). Scale bar: 20 μm. (b) Quantitative analysis of ZIKV E-positive 293T cells from (a). (c) Fluorescence intensity profile along white arrows in (a), indicating sites of ITGB2 and ZIKV E co-localization. (d) 293T cells were transfected with increasing amounts (0.5, 1, 2 μg) of ITGB2 plasmid and infected with ZIKV (MOI = 1) for 1 h. ZIKV RNA levels were measured by qPCR to assess infection susceptibility (mean ± SD, n = 3). (e) Molecular docking model predicting the interaction interface between ITGB2 and ZIKV E protein. (f) Co-IP assay in 293T cells, followed by immunoblotting with anti-ZIKV E protein antibody. (g) CBNK-EVs were pre-incubated with ITGB2 mAb (0, 5, 20 μg/mL) before being applied to Vero-E6 or BHK-21 cells. Cells were then infected with ZIKV (MOI = 1) for 1 h, and antiviral activity was assessed by measuring ZIKV RNA levels at 24 h post-infection. Data are presented as mean ± SD (n = 3 for d and g; n = 6 for b). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 (one-way ANOVA).

Article Snippet: Vero E6, BHK21, 293T and HTR-8/Svneo cells were purchased from ATCC.

Techniques: Binding Assay, Multicolor Immunofluorescence Staining, Transfection, Fluorescence, Plasmid Preparation, Infection, Co-Immunoprecipitation Assay, Western Blot, Incubation, Activity Assay

Journal: The Journal of Experimental Medicine

Article Title: DNA-PK interacts with cyclic dinucleotides and inhibits type I interferon responses

doi: 10.1084/jem.20251796

Figure Lengend Snippet: 2′3′-cGAMP interacts with the catalytic pocket of DNA-PKcs. (A) Experimental scheme for B and C. FLAG-tagged DNA-PKcs (F-DNA-PKcs or FLAG-DNA-PKcs) expressed in 293T cells was FLAG was subjected to immunoprecipitation (IP), prior to incubation with 2′3'-cGAMP, release of bound 2′3′-cGAMP, and detection by ELISA. (B) WB analysis of input and FLAG-IP performed as in A was conducted using the indicated antibodies. Representative WB of three independent experiments. (C) 2′3′-cGAMP was measured by ELISA on experiment performed as in A. Graph presents the mean ± SEM of three independent experiments. Statistical significance was calculated by two-tailed Student's t test. (D) Experimental scheme for E. Recombinant DNA-PKcs was immunoprecipitated using a DNA-PKcs–specific antibody, prior to incubation with 2′3′-cGAMP, release of bound 2′3′-cGAMP, and detection by ELISA. (E) Graph represents mean (±SEM) 2′3′-cGAMP levels as measured in mock IgG and DNA-PK–specific IP performed as in D. Statistical significance was calculated by two-tailed Student's t test. n = 3 independent experiments. (F) Experimental scheme for G. FLAG-tagged DNA-PKcs (FLAG-DNA-PKcs) expressed in 293T cells was FLAG purified prior to incubation with biotin or biotinylated 2′3′-cGAMP (C3-2′3′-cGAMP), followed by streptavidin pull-down and WB analysis. (G) WB analysis of input and streptavidin pull-down experiment performed as in F was conducted using a FLAG-specific antibody. Representative WB of three independent experiments. (H) DNA-PKcs (red) and 2′3′-cGAMP (green) subcellular localization was assessed 6 h after iFluor488-2′3′-cGAMP transfection in T98G cells. Immunofluorescence was performed using a DNA-PKcs–specific antibody and DAPI nuclear staining. Representative images of 15–20 images. Scale bars, 5 µm. (I) Quantification of cytosolic DNA-PKcs and iFluor488-2′3′-cGAMP foci colocalization following transfection of T98G cells with mock or fluorescent 2′3′-cGAMP using the CellProfiler software. n = 424 and 558. Statistical significance was calculated by two-tailed Student's t test. (J) Experimental scheme for K. THP-1 cells were processed for TSA in the presence or absence of 2′3′-cGAMP. (K) WB analysis of TSA, as described in J, was conducted using indicated antibodies. Representative WB of three independent experiments. (L) Experimental scheme for M. Purified FLAG-DNA-PKcs was used as input material for TSA in the presence or absence of 2′3′-cGAMP. (M) WB analysis of TSA, performed as in L, was conducted using anti-FLAG antibody. Representative WB of three independent experiments. (N) Representation of the molecular modelling of 2′3′-cGAMP in interaction with DNA-PKcs. (O) ATP hydrolysis by DNA-PK was measured in vitro in presence of NU7441 or increasing doses (300–2,700 µM) of 2′3′-cGAMP. Graph presents the mean of three independent experiments. One-way ANOVA. (P) As in D, except that DNA-PKcs IP was incubated with or without 2′3′-cGAMP in presence or absence of NU7441 (used as a competitor) prior to measurement of bound 2′3′-cGAMP. Graph represents mean (±SEM) 2′3′-cGAMP levels; n = 3 independent experiments. Statistical significance was calculated by two-tailed Student's t test. (Q) FLAG-DNA-PKcs, FLAG-DNA-PKcs-Δkinase, and FLAG-kinase were expressed in 293T cells prior to TSA analysis in the presence or absence of 2′3′-cGAMP. WB was conducted with the indicated antibodies. Representative WB of three independent experiments. (R) FLAG-DNA-PKcs, FLAG-DNA-PKcs-Δkinase, and FLAG-kinase were FLAG purified as in A prior to incubation with biotin or biotinylated 2′3′-cGAMP and binding analysis by WB as in G using FLAG antibody. Representative WB of three independent experiments. ***: P < 0.001; **: P < 0.01; *: P < 0.05; ns, not significant. Also see . Source data are available for this figure: .

Article Snippet: To generate the T98G IFNAR−/− knockout and control cell lines, lentiviral particles were produced by co-transfection of 2 × 10 6 293T cells with 5 μg of LentiCRISPRv2GFP plasmid (#82416; Addgene) expressing the gRNA targeting the gene of interest or nontargeting control gRNA, 5 μg of psPAX2, and 1 μg of pMD2.G, using the standard calcium phosphate transfection protocol.

Techniques: Immunoprecipitation, Incubation, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Recombinant, Purification, Transfection, Immunofluorescence, Staining, Software, In Vitro, Binding Assay