Figure 3

Detection of viral gene expression in xenotransplanted cells by ddPCR: (A) TREx-BCBL1-RTA cells reactivated with 1 µg/mL of doxycycline in culture and RNA was harvested at latent cells, or cells undergoing lytic replication at 24 or 48 h post-induction (hpi). Then, 500 µM phosphonoacetic acid (PAA) was used to inhibit replication of the viral genome and late gene expression; RT-qPCR was used to measure transcript abundance of β-actin, RTA (immediate early), ORF45 (early) and K8.1 (late) (n = 4 independent experiments; means ± SEM); (B) Western blot of cells treated as in (A) to confirm accumulation of target proteins; (C) ddPCR amplification plot for β-actin, RTA, ORF45, and K8.1. The x axis displays individual events, and the y axis is fluorescence amplitude. For all targets, we tested cDNA generated from uninjected larvae, or larvae injected with untreated TREx-BCBL1-RTA cells or cells treated with 1 µg/mL doxycycline for 12 h prior to injection. RNA was harvested from larvae at 48 hpi. The pink threshold line separates positive reaction droplets (blue) from negative droplets (gray).