Proliferation and lytic reactivation of iSLK.219 in zebrafish embryos: (A) simplified diagram of rKSHV.219 reporter construct, adapted from [17,19]. Latently infected cells express GFP from a constitutive EF-1α promoter. During lytic replication, the immediate early protein RTA binds to the viral PAN promoter and stimulates RFP expression. A polyA (pA) signal sequence is present on both strands of the viral genome; (B) proliferation of iSLK.219 cells at 2 and 3 dpi, normalized to the number of cells counted at 1 dpi (n = 3 independent experiments with cells from 20 larvae counted per measurement; means ± SEM; statistical significance was determined by two-way ANOVA compared to the cell counts at 1 dpf; * = p < 0.05); (C) iSLK.219 cells were treated with 1 µg/mL of doxycycline and fixed at the times indicated, or left untreated (Time = 0 hpi). Cells were fixed with 4% paraformaldehyde, and nuclei were stained with Hoescht. RFP+ cells and nuclei were imaged on an inverted fluorescent microscope and enumerated with CellProfiler (n = 3 independent experiments ± SD; nd = not detected). (D) iSLK.219 were injected into the yolk sac of 2 dpf zebrafish embryos. The following days, larvae were screened for viability and a GFP+ cell bolus by fluorescence microscopy. In half of the larvae, the E3 media was supplemented with 40 µg/mL doxycycline, which was refreshed daily. Xenotransplanted larvae were monitored daily for RFP+ cells. Presented here are representative images of both doxycycline-activated and mock-treated larvae at the ethical endpoint of the experiment. We could observe RFP+ cells in the yolk sac of approximately 20% of larvae treated with doxycycline, and none in untreated larvae (scale bar = 100 µm).
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