Journal: Virulence

Article Title: Streptococcus mutans -derived extracellular vesicles promote herpes simplex virus infection in oral epithelia

doi: 10.1080/21505594.2025.2602261

Figure Lengend Snippet: Effect of Sm EVs on HSV-1 infection in HOK cells. (A) Confocal imaging of HOK cells treated with Sm EVs labeled with Nile red for 3 h. HOK cells were stained with phalloidin (green), and nuclei were stained with DAPI (blue). Fluorescence was captured using a ZEISS LSM800 confocal microscope with Airyscan. (B) HOK cells were treated with Sm EVs at concentrations ranging from 0 particles/cell to 1 × 10 4 particles/cell for 48 h. Cell viability was assessed using an MTT assay. (C) HOK cells were pretreated with Sm EVs at a concentration of 1 × 10 3 particles/cell for 24 h. Subsequently, the cells were infected with HSV-1 at MOIs of 0.01, 0.1, and 1 for an additional 48 h. A mock-infected group (MOI 0) was included as a control. The cytotoxic effect of HSV-1 was assessed using an MTT assay. (D) After 48 h post-infection, viral rna was extracted from culture supernatants collected from the HSV-1-infected HOK cells, and gD mRNA levels were quantified using qPCR. (E) Representative plaque assay images and quantification in HOK cells pretreated with or without Sm EVs. For plaque assays, supernatants from infected HOK cells were collected, serially diluted, and applied to monolayers of Vero cells. After viral adsorption, cells were overlaid with agarose and incubated until plaques formed, which were then visualized and counted to determine PFU. Results are presented as the mean standard deviation of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, and ns, not significant.

Article Snippet: HOK cells were cultured in keratinocyte growth medium 2 (PromoCell, Heidelberg, Germany) supplemented with a supplement mix and CaCl 2 solution.

Techniques: Infection, Imaging, Labeling, Staining, Fluorescence, Microscopy, MTT Assay, Concentration Assay, Control, Plaque Assay, Adsorption, Incubation, Standard Deviation

Journal: Virulence

Article Title: Streptococcus mutans -derived extracellular vesicles promote herpes simplex virus infection in oral epithelia

doi: 10.1080/21505594.2025.2602261

Figure Lengend Snippet: Effect of the EGFR – ERK pathway on the Sm-EV-dependent increase of HSV-1 production in HOK cells. (A) Cells were pretreated with EGFR antagonist (AG1478) for 3 h and then treated with Sm EVs for 24 h. After infection with HSV-1 (MOI 0.1) for another 48 h, the cells were stained with fluorescent gD antibody. Fluorescence intensity was quantified using zen microscopy software. (B) The protein level of HSV-1 gD was analyzed using western blot analysis under the same conditions. EGFR, erk, and NF-κB phosphorylation levels were analyzed using western blot analysis under the same conditions. (C) Cells were pretreated with erk antagonist (PD98059) for 3 h and then treated with Sm EVs for 24 h. After infection with HSV-1 (MOI 0.1) for another 48 h, western blot analysis was performed to measure gD protein levels and erk phosphorylation. β-actin was used as an internal loading control. (D) Cells were pretreated with AG1478 for 3 h and then treated with Sm EVs for 24 h. After infection with HSV-1 (MOI 0.1) for another 48 h, qPCR was performed to measure IFN mRNA level. (E) Cytokine ELISA was performed to measure the levels of secreted IFN proteins under the same conditions. Results are presented as the mean ± standard deviation of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: HOK cells were cultured in keratinocyte growth medium 2 (PromoCell, Heidelberg, Germany) supplemented with a supplement mix and CaCl 2 solution.

Techniques: Infection, Staining, Fluorescence, Microscopy, Software, Western Blot, Phospho-proteomics, Control, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Virulence

Article Title: Streptococcus mutans -derived extracellular vesicles promote herpes simplex virus infection in oral epithelia

doi: 10.1080/21505594.2025.2602261

Figure Lengend Snippet: Quantification of HSV-1 production by plaque assay following Sm EV treatment and EGFR – ERK inhibition. (A) HOK cells were pretreated with Sm EVs (1 × 10 3 particles/cell) for 24 h, followed by HSV-1 infection (MOI 0.1) under the indicated conditions: non-treated (NT), AG1478 (10 μM), or PD98059 (20 μM). After 48 h, culture supernatants were collected, and HSV-1 titers were determined by plaque assay. (B) Representative plaque images from culture supernatants. Results are presented as the mean ± standard deviation of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: HOK cells were cultured in keratinocyte growth medium 2 (PromoCell, Heidelberg, Germany) supplemented with a supplement mix and CaCl 2 solution.

Techniques: Plaque Assay, Inhibition, Infection, Standard Deviation