Transfection of COS-7 cells with <i>in vitro</i> transcribed ssRNAs encoding RV ssRNAs and DLP derived ssRNAs.

<p>COS-7 cells at 80% confluence were transfected with ssRNAs encoding RV proteins using Mirus transfection reagent. Cells were fixed at 24 hours post transfection and stained with NSP2 and NSP5-specific antibodies (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074328#pone.0074328.s012" target="_blank">Table S4</a>). Images were analysed by confocal microscopy. Panel A: transfection with cohorts of <i>in vitro</i> transcribed ssRNAs, 1 µg of S1– S11, post-capped (PC) or co-capped (CC) respectively, stained for both NSP2 and NSP5. Panel B: 1 µg of DLP derived ssRNAs from RV strains RF, 125 and 128 respectively, stained for both NSP2 and NSP5. Panel C: individual or co-transfection of 500 ng of ssRNAs S8, S11 or both stained for NSP2, NSP5 or both, respectively. Panel D: immunofluorescence of control MA104 cells infected with RF RV, stained for NSP2 and NSP5, transfection control with 1 µg of eGFP ssRNAs (autofluorescence), mock transfection stained for NSP2 and NSP5. Cell nuclei were stained with Hoechst 33342 in all panels. Scale bars: 20 µm.