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. 2022 Feb 9;14(2):354. doi: 10.3390/v14020354

Figure 4.

Figure 4

Diphyllin 1 mechanism of action assessment and comparison of biological activity of diphyllin 1 with that of bafilomycin A1. (A) Analysis of endosomal acidification with acridine orange labeling. Vero cells cultured in 96-well microtiter plates were treated with diphyllin 1, bafilomycin A1, or DMSO (1%, v/v) (controls) at the indicated concentrations at 37 °C for 20 min and then incubated with acridine orange dye (1 μg/mL). Scale bars, 50 µm. (B) Cytotoxicity of bafilomycin A1 for Vero cells in the indicated concentration range. The cells were seeded in 96-well plates for 24 h, then treated with bafilomycin A1 and incubated for 48 h. (C) Anti-ZIKV and anti-HSV-1 activities of bafilomycin A1 in Vero cells. Vero cell monolayers were treated with bafilomycin A1 and simultaneously inoculated with ZIKV or HSV-1 (MOI of 0.1). The infected cells were then incubated for 48 h, after which cell media were collected, and viral titers were determined using a plaque assay. (D) Anti-ZIKV and anti-HSV-1 activities of diphyllin 1 (100 µM) and bafilomycin A1 (100 nM) in Vero cells measured after 3 h p.i. DMSO-treated cells were used as controls. Vero cell monolayers inoculated with the indicated viruses and diphyllin 1 (100 µM), bafilomycin A1 (100 nM), or DMSO (1%, v/v) were added to the cells for 3 h at 37 °C. After extensive washing, fresh medium with 4% (w/v) carboxymethylcellulose was added to the cell monolayers. After 72 h (for HSV-1) or 120 h (for ZIKV), the cells were stained with naphthalene black, and the number of plaques was determined. (E) Control cells were inoculated with the appropriate viruses and incubated with the compounds for 72 or 120 h for HSV-1 and ZIKV, respectively. Then, the cells were stained with naphthalene black, and the number of plaques was determined. (F,G) ZIKV and HSV-1-infected Vero cell monolayers in 6-well plates treated with the indicated compounds. The experimental conditions were described above, see (D,E). The horizontal dashed line represents 90% threshold for compound cytotoxicity assessment. MI: MacIntyre; N.D.: not detected.