Glass coverslips

Overnight subconfluent monolayers of Ohio-HeLa or A549 cells grown on glass coverslips (Proscitech, #1) were infected with HRV16 or HRV2 at an MOI of 1 (Ohio-HeLa cells) or 5 (A549 cells) or left uninfected (mock) and fixed with 4% formaldehyde in PBS followed by permeabilization of cell membranes with 0.2% Triton X-100 in PBS at various times post infection (p.i.) (Ghildyal et al., 2005 (link)). Cells were incubated with primary antibodies diluted in PBS for 30 min, washed twice in PBS, incubated with species specific secondary antibodies conjugated to CF 488 (Biotium) or Alexa Fluor 568 diluted 1:1000 in PBS for 30 min, and then washed twice in PBS and mounted using ProLong Gold mounting media with DAPI (Invitrogen). Digitized fluorescent cell images were collected using a Nikon Ti Eclipse confocal laser-scanning microscope (CLSM) with Nikon 60×/1.40 oil immersion lens (Plan Apo VC OFN25 DIC N2; optical section of 0.5 μm) and the NIS Elements AR software. Quantitative analysis of the fluorescence signal in the nucleus (Fn) and cytoplasm (Fc) was performed using ImageJ.

Single-molecule spectroscopy was performed as previously described [9 (link)]. Briefly, samples (20 μl) were loaded into a custom-made silicone 192-well plate adhered to glass coverslips (ProSciTech Australia). Samples were analysed with two lasers (488 nm and 561 nm) using a Zeiss LSM710 microscope with a Conforcor3 module for single-molecule counting and a single 488-nm laser for aggregation analyses. The fluorescence emission was filtered with 505–540-nm band pass filter (GFP) and 580-nm long-pass filter (mCherry). Measurements were taken with photon counts in the approximate range of 750–2,000 which corresponds to a GFP concentration of around 1–2.5 μg/ml. Three replicates were carried out for each construct pair, and consistent results were obtained for each.