Fig. 3. 4′-FlU is efficiently anabolized in HAE cells and is efficacious in human airway epithelium organoids.
(A to C) 4′-FlU cellular uptake and metabolism in “F1” HAE cells quantified by mass spectrometry (A). Intracellular concentration of 4′-FlU(-TP) after exposure to 20 μM 4′-FlU for 0, 1, 2, 3, 4, 6, 16, and 24 hours (B), or 24-hour incubation followed by removal of the compound for 0, 0.5, 1, 2, 3, and 6 hours before quantification (C) (n = 3). The low limit of quantitation (LLOQ) for 4′FlU (19.83 pmol/106 cells) is indicated by the dashed line. (D) HAE cells were matured at air-liquid interface (ALI). (E) Virus yield reduction of recRSV-A2line19F-[FireSMASh] was shed from the apical side in ALI HAE after incubation with serial dilutions of 4′-FlU on the basal side (n = 3). (F to H) Confocal microscopy of ALI HAE cells infected with recRSV-A2line19F-[FireSMASh], at 5 days after infection. RSV infected cells, tight junctions, and nuclei were stained with anti-RSV, anti-ZO-1, and Hoechst 34580. z-stacks of 30 × 1 μm slices with 63 × oil objective. Dotted lines, x-z and y-z stacks; scale bar, 20 μm. In all panels, symbols represent independent biological repeats and lines represent means.