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. 2022 May 12;185(12):2086–2102.e22. doi: 10.1016/j.cell.2022.04.022

Figure S4.

Figure S4

FD duplexes in the context of HSV-1 infection, related to Figure 3

(A) Flow cytometry dot plots of naive or FD175-nucleofected ARPE-19 cells after infection with HSV-1 strain 17syn+ IE175-YFP (MOI = 0.1). Cells were infected 24 h post-nucleofection with FD175 and analyzed at 4 hpi. The uninfected control is the same as in Figure S3A.

(B) Fluorescent micrographs of Vero cells (nucleofected ± FD175), then infected with HSV-1 17syn+ IE175-YFP, imaged at 1 hpi (MOI = 20), and stained for ICP5 protein (red, 594 nm) and DAPI (blue, 405 nm).

(C) Flow cytometry live-dead analysis using Zombie Aqua of uninfected (naive) Vero cells (i.e., control) showing gating for dead cells. Dead cell gate was drawn by comparing with HSV-1-infected Vero cells in Figure S5D (left-most column). Percentage of dead cells is <1%.

(D) Uninfected Vero cells nucleofected with FDScram or FD175 (mean of biological triplicates shown); nucleofection of FD175 does not generate significant increases in dead cells compared with nucleofection of FDScram control.

(E) FD175 interferes with HSV-1 (KOS strain) replication. Vero cells were nucleofected with either 25 μM FD175 (or mock/FDScram) and 24 h later were infected with HSV-1 (KOS strain) at MOI = 0.1, then virus quantified by TCID-50 at 4 dpi.

(F and G) Absence of nonspecific antiviral effects. (F) Vero cells were nucleofected with 25 μM FD86 or FDScram and infected with HSV-1 (17syn+ strain IE175-YFP virus, MOI = 0.1), followed by quantification of virus titer 4 dpi by TCID50. (G) ARPE-19 cells were nucleofected with 25 μM FD86 or FDScram and infected with CMV (TB40E-IE86-YFP MOI = 0.1), followed by quantification of virus titer 4 dpi by TCID50.

(H) Left: flow cytometry plots of naive Vero cells or in the presence of 100 μM FD175 stained with Zombie Aqua stain at 4 dpi; right: quantification of % Zombie Aqua positive cells from the flow plots.

(I) Left: flow cytometry plots of naive Vero cells or in the presence of 100 μM FD175 + 10μM of acyclovir stained with Zombie Aqua stain at 4 dpi; right: quantification of % Zombie Aqua positive cells from the flow plots.