hela  (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: Characterization and anti-ZIKV activity of CBNK-EVs. (a) Isolation of CBNK cells by fluorescence-activated cell sorting. (b) Collection of CBNK-EVs and NTA for size distribution and concentration. (c) Morphological observation of CBNK-EVs by transmission electron microscopy, scale bar:100 nm. (d) WB analysis of specific markers for CBNK-EVs. (e) Anti-ZIKV activity of CBNK-EVs assessed by immunofluorescence. Vero-E6 cells were seeded in 12-well plates at 1 × 10 5 cells per well one day prior to experiments. Cells were pretreated with varying volumes of CBNK-EVs (stock concentration: 3 × 10 11 particles/mL) for 1 h to achieve the indicated final particle counts, followed by infection with ZIKV (MOI = 1) for 24 h in the presence of fresh medium containing the same EVs. ZIKV-infected cells were quantified by counting E protein-positive cells in three random fields by two independent investigators. Scale bar, 40 μm. (f) Statistical analysis of the data presented in (e) panel. (g) Vero-E6 cells were treated as in (e) and infected with ZIKV (MOI = 1) for 24 h. (h) ZIKV E and NS1 RNA levels were detected by quantitative RT-PCR. Data were normalized to GAPDH, log10-transformed, and presented as fold change compared to the virus control group (set to 1). Negative values indicate inhibition of viral replication. (i) Plaque reduction assay assessing the antiviral activity of CBNK-EVs against ZIKV. (j) Statistical analysis of the percentage of plaque reduction. (k) ZIKV NS5 protein levels in (g) were analyzed by Western blot. Data in panels (f, h, j and k) are presented as mean ± SD (n = 3). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

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

Techniques: Activity Assay, Isolation, Fluorescence, FACS, Concentration Assay, Transmission Assay, Electron Microscopy, Immunofluorescence, Infection, Quantitative RT-PCR, Transformation Assay, Virus, Control, Inhibition, Western Blot

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: Functional components and mechanisms of CBNK-EVs in combating ZIKV infection. (a) Schematic of three treatment modalities: 1) pre-treatment of cells with CBNK-EVs, 2) pre-treatment of virus with CBNK-EVs, and 3) co-incubation of CBNK-EVs with ZIKV. (b) Vero-E6 cells were seeded at 1 × 10 5 cells per well in 12-well plates and subjected to the treatments outlined in (a), followed by infection with ZIKV (MOI = 1) for 1 h. After 48 h, ZIKV E and ZIKV NS5 protein levels were analyzed by Western blot (left). Densitometric analysis of the protein bands is shown (right, n = 3). (c) Schematic workflow for functional component inactivation in CBNK-EVs (e.g., by heat, protease, or nuclease treatment). (d) Vero-E6 cells were treated with inactivated CBNK-EVs as per the scheme in (c), followed by infection with ZIKV (MOI = 1) for 1 h. ZIKV E and ZIKV NS5 protein levels were analyzed by Western blot after 24 h. Densitometric analysis of the protein bands is shown (right, n = 3). (e) Represent images of the co-localization of CBNK-EVs, ZIKV E and host cells by confocal microscope, scale bar: 5 μm. (f) Fluorescence intensity of CBNK-EV and β-actin co-localization signals (white arrows) in the EVs group. (g) Fluorescence co-localization signals (yellow arrows) of CBNK-EVs and ZIKV in the co-culture group. (h) TEM images of CBNK-EVs, ZIKV particles, and their interactions. Scale bar: 100 nm. (i) Membrane disruption assay. ZIKV particles were co-incubated with CBNK-EVs, followed by treatment with micrococcal nuclease to digest unprotected RNA. Intact viral RNA, indicative of membrane integrity, was then measured by RT-qPCR targeting the ZIKV E. Mean ± SD (n = 3). ∗∗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: Functional Assay, Infection, Virus, Incubation, Western Blot, Microscopy, Fluorescence, Co-Culture Assay, Membrane, Disruption, Quantitative RT-PCR

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 Cell Surface

Article Title: Pap1 is an adhesin involved in the interaction of Sporothrix schenckii and Sporothrix brasiliensis with the host

doi: 10.1016/j.tcsw.2025.100164

Figure Lengend Snippet: PAP1 expression under different growing conditions . Yeast-like cells were incubated under the following conditions at 37 °C: 1 h with either 1.0 μg mL −1 fibronection or thrombospondin 1; 1 h with a monolayer of HeLa cells; 1 h with 5 × 10 6 human PBMCs, or injected in the hemolymph of Galleria mellonella larvae and incubated for 24 h. Alternatively, biofilms were matured for 48 h at 37 °C. From these conditions, total RNA was extracted, cDNA synthesized with oligo(dT) primer (20 mer), and PAP1 expression quantified by RT-qPCR. Data were normalized using the expression of the gene encoding the ribosomal protein L6 and yeast-like cells growth in YPD at 37 °C as reference conditions (point zero on the Y axis). Results are means ± SD of three independent experiments performed in duplicate. The Dunnett's test and then the unpaired t -test were used for data analysis. * P < 0.05 when compared to the other growing conditions.

Article Snippet: In some experiments, HeLa cells (ATCC) were grown in monolayers in Eagle's Minimum Essential Medium (EMEM, Sigma-Aldrich) at 37 °C and 5 % CO 2 (v/v).

Techniques: Expressing, Incubation, Injection, Synthesized, Quantitative RT-PCR

Journal: The Cell Surface

Article Title: Pap1 is an adhesin involved in the interaction of Sporothrix schenckii and Sporothrix brasiliensis with the host

doi: 10.1016/j.tcsw.2025.100164

Figure Lengend Snippet: Adhesion to extracellular matrix components and HeLa cells of Sporothrix schenckii and Sporothrix brasiliensis PAP1 -silenced strains. The indicated extracellular matrix component was used to coat 96-well plates, then yeast-like cells were added, unbound cells were removed by extensive washing, and adherent cells were detected by ELISA with a primary anti-rHsp60 antibody. Control refers to wells coated with bovine serum albumin. Panel A contains data generated with S. schenckii, and WT is the 1099–18 ATCC MYA 4821 strain. Panel B shows results with S. brasiliensis cells, and the WT is the 5110 ATCC MYA 4823 strain. Alternatively, for both panels, 1 × 10 6 HeLa cells were placed per well, incubated 24 h at 37 °C and 5 % ( v /v) CO 2 , and used in the adhesion assays. Results are means ± SD of three biological replicates performed in duplicate. The Dunnett's test and then the unpaired t-test were used for data analysis. In both panels, * P < 0.05 when compared to WT or strains HSB1 and HSB2.

Article Snippet: In some experiments, HeLa cells (ATCC) were grown in monolayers in Eagle's Minimum Essential Medium (EMEM, Sigma-Aldrich) at 37 °C and 5 % CO 2 (v/v).

Techniques: Enzyme-linked Immunosorbent Assay, Control, Generated, Incubation

Journal: Bioactive Materials

Article Title: Multimodal profiling of CAR T cells against glioblastoma using a microengineered 3D tumor-on-a-chip model

doi: 10.1016/j.bioactmat.2026.01.003

Figure Lengend Snippet: Off-target cytotoxicity evaluation of CAR T cells using the 3D GOC system. A) Schematic representation of the differing cytolytic mechanisms of UTD, TV-13, and IL-13 CAR T cells against IL13Rα1 + HT-1080 tumor cells. Created with BioRender.com . B) Flow cytometric analysis confirming IL13Rα1 and mCherry (reporter gene) expression on IL13Rα1 + HT-1080 tumor cells. Antigen expression (IL13Rα1 or mCherry) on viable tumor cells shown in histograms: blue for IL13Rα1 + HT-1080 tumor cells and red for control tumor cells. The values within each histogram indicate the percentage of positive cells, with the mean fluorescence intensity (MFI) shown in parentheses. C) Microfluidic evaluation of off-target toxicities of T cells. (i) Representative tile images of tumor-stroma interface stained for actin cytoskeleton (green), showing differences in migration of IL13R1 + HT-1080 tumor cells (red) within the 3D GOC model across varying densities of UTD, TV-13 CAR, and IL-13 CAR T cells. (ii) Quantification of the migration distance of the IL13Rα1 + HT-1080 tumor cells in response to varying T cell concentrations. Data are represented as mean ± SD measured from three biological replicates ( n = 3) , T cell donors: DN18, DN28, and DN31, ∗ p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗ ∗p < 0.0001. Two-way ANOVA with Tukey's multiple comparisons test was utilized for statistical analysis. (iii) Bar graph showing the difference in nuclei per field of view (FOV) across different T cell densities, used as a measure of chain migration by IL13Rα1 + HT-1080 tumor cells. Data are represented as mean ± SD measured from three biological replicates ( n = 3) , T cell donors: DN18, DN28, and DN31, ∗ p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Two-way ANOVA with Tukey's multiple comparisons test was utilized for statistical analysis, and (iv) Bar graph representing the percentage of T cells positive for intracellular cytokines in the presence of IL13Rα1 + HT-1080 tumor cells. Data are represented as mean ± SD measured from three biological replicates ( n = 3) , ∗ p < 0.05, ∗∗p < 0.01, ∗∗∗ ∗p < 0.0001. Two-way ANOVA with Tukey's multiple comparisons test was utilized for statistical analysis.

Article Snippet: HT-1080 Culture : Human fibrosarcoma cells (CCL-121, ATCC or HT-1080) were used to generate an off-target cell line (IL13Rα1 + HT-1080) expressing IL13Rα1-T2A-mCherry gene, which was single-sorted for the experiments described here.

Techniques: Gene Expression, Expressing, Control, Fluorescence, Staining, Migration

Journal: Bioactive Materials

Article Title: Multimodal profiling of CAR T cells against glioblastoma using a microengineered 3D tumor-on-a-chip model

doi: 10.1016/j.bioactmat.2026.01.003

Figure Lengend Snippet: Off-target cytotoxicity evaluation of CAR T cells using the 3D GOC system. A) Schematic representation of the differing cytolytic mechanisms of UTD, TV-13, and IL-13 CAR T cells against IL13Rα1 + HT-1080 tumor cells. Created with BioRender.com . B) Flow cytometric analysis confirming IL13Rα1 and mCherry (reporter gene) expression on IL13Rα1 + HT-1080 tumor cells. Antigen expression (IL13Rα1 or mCherry) on viable tumor cells shown in histograms: blue for IL13Rα1 + HT-1080 tumor cells and red for control tumor cells. The values within each histogram indicate the percentage of positive cells, with the mean fluorescence intensity (MFI) shown in parentheses. C) Microfluidic evaluation of off-target toxicities of T cells. (i) Representative tile images of tumor-stroma interface stained for actin cytoskeleton (green), showing differences in migration of IL13R1 + HT-1080 tumor cells (red) within the 3D GOC model across varying densities of UTD, TV-13 CAR, and IL-13 CAR T cells. (ii) Quantification of the migration distance of the IL13Rα1 + HT-1080 tumor cells in response to varying T cell concentrations. Data are represented as mean ± SD measured from three biological replicates ( n = 3) , T cell donors: DN18, DN28, and DN31, ∗ p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗ ∗p < 0.0001. Two-way ANOVA with Tukey's multiple comparisons test was utilized for statistical analysis. (iii) Bar graph showing the difference in nuclei per field of view (FOV) across different T cell densities, used as a measure of chain migration by IL13Rα1 + HT-1080 tumor cells. Data are represented as mean ± SD measured from three biological replicates ( n = 3) , T cell donors: DN18, DN28, and DN31, ∗ p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Two-way ANOVA with Tukey's multiple comparisons test was utilized for statistical analysis, and (iv) Bar graph representing the percentage of T cells positive for intracellular cytokines in the presence of IL13Rα1 + HT-1080 tumor cells. Data are represented as mean ± SD measured from three biological replicates ( n = 3) , ∗ p < 0.05, ∗∗p < 0.01, ∗∗∗ ∗p < 0.0001. Two-way ANOVA with Tukey's multiple comparisons test was utilized for statistical analysis.

Article Snippet: HT-1080 Culture : Human fibrosarcoma cells (CCL-121, ATCC or HT-1080) were used to generate an off-target cell line (IL13Rα1 + HT-1080) expressing IL13Rα1-T2A-mCherry gene, which was single-sorted for the experiments described here.

Techniques: Gene Expression, Expressing, Control, Fluorescence, Staining, Migration