Eclipse ti2 system

To verify the expression of recombinant proteins in human cells, cells were seeded on 12 mm cover slips. After transfection, cells were fixed in 4% paraformaldehyde (PFA) in PBS, pH 7.4 at room temperature for 15 min. Next, cells were permeabilized with 0.5% Triton in PBS for 5 min and blocked in Roti Immunoblock (Roth) at 4°C overnight. To stain HA (Hemagglutinin peptide)-tagged or EYFP (Enhanced Yellow Fluorescent Protein)-tagged proteins, respectively, the fixed cells were incubated for 1 h in a 1:500 diluted solution of primary antibody (α-HA rabbit, abcam ab9110, or monoclonal α-GFP mouse, Roth, respectively), washed in PBS and afterwards incubated for 1 h in a 1:1000 diluted Alexa594 labelled secondary antibody (Thermo Fisher) solution and DAPI (4,6-diamidino-2-phenylindole). After final washing, the cells were mounted on microscope slides using Fluoromont-G mounting medium (Southernbiotech). The localization of EYFP- and HA-tagged PPR proteins was examined on a Nikon Eclipse Ti2 system, equipped with a PlanFluor 40× Oil objective (NA 1.3) using the NIS-Elements AR software and ImageJ/Fiji version 1.53c for Windows. Transfection rates were calculated using Fiji as outlined in Supplementary Figure S1.

MDCK cells (90% confluent in 96-well plates) were infected and, after 1 h at 37 °C, the virus inoculum was removed and the cells were washed with PBS and overlaid with 1% methylcellulose in Modified Eagle’s Medium. At 16 h post-infection at 37 °C, the cells were fixed with 4% paraformaldehyde in PBS, incubated with anti-H5N1 virus polyclonal antibody for 1 h at 37 °C, washed 3 times with PBS, and reacted with Alexa Fluor 488 anti-rabbit antibody for 1 h at 37 °C. After 3 washes with PBS, the plates were coated with a 50% glycerol solution. Digital images were taken using an inverted fluorescence microscope ECLIPSE Ti2 system (Nikon) and foci sizes were measured using NIS-Elements software (Nikon).