Extended Data Fig. 8: Examination of RSV entry fusion and colocalization using RSV–DiD on 1HAEo⁻ cells expressing NCL–GFP.

a, 1HAEo⁻ cells were infected with DiD-labelled RSV (red) on ice, fixed, stained for RSV-F (green) and imaged using fluorescence microscopy. b, Immunoprecipitation using an anti-RSV-F antibody of RSV-infected HEK-293T cells transiently expressing either NCL–GFP or GFP. NCL (left) and GFP (right) were detected by western blot. c, d, Living 1HAEo⁻ cells expressing NCL–GFP (c) or GFP alone (d) were infected with RSV-DiD (red) and imaged over time (the time (in min) after infection is indicated above the panels). Arrowheads denote fusing RSV particles, as indicated by an increase in DiD fluorescence (see schematic in Fig. 2h). DiD–RSV was used in Fig. 2c, i, j, and Supplementary Videos 1, 2. All RSV infections in this figure were synchronized on ice for 1 h before warming, t = 0 represents the time at which the cells are transferred to 37 °C from ice. Selected images are representative of multiple fusion events in at least 4 independent experimental repeats.