Figure 6.

Translation and visualization of tC^O-labeled mRNA in human SH-SY5Y cells. The mRNA translation was monitored based on the fluorescence intensity of the encoded H2B:GFP protein using confocal microscopy and flow cytometry. (a) mRNA translation following electroporation; (top) confocal images (3× enlargements, scale bars: 10 μm) recorded 24 h postelectroporation, (middle) flow cytometry scatter plots 24 h postelectroporation, and (bottom) mean cellular GFP fluorescence intensity (MFI GFP ± standard deviation) of all counted cells at 24, 48, and 72 h postelectroporation. (b) Representative MFI GFP histograms corresponding to the distributions in (a). (c) mRNA translation following chemical transfection (lipofection), corresponding to the data shown in (a). (Top) Confocal images (3× enlargements, scale bars: 10 μm) recorded 48 h postchemical transfection, (middle) flow cytometry scatter plots 48 h postelectroporation, and (bottom) MFI GFP of all counted cells at 24, 48, and 72 h postchemical transfection. (d) Snap-shot images from a confocal time-lapse experiment to monitor the intracellular trafficking of 75% tC^O-labeled mRNA (red) introduced, by chemical transfection, into cells with an overexpression of mRFP-Rab5 to label early endosomes (orange). Resulting expression of H2B-GFP protein in the nucleus is shown in green. White arrows indicate discrete mRNA–lipid complexes; scale bars: 10 μm. Supplementary Movie 1 shows the full time lapse. (e) mRNA translation of cyanine5-labeled (Cy5) eGFP encoding mRNAs (TriLink) 24 h post-transfection (electroporation or chemical transfection). NL; nonlabeled. Scale bars: 10 μm. (f) Impact of tC^O or Cy5 incorporation on mRNA translation, represented as the ratio of cellular MFI GFP of the labeled mRNA relative to the cellular MFI GFP for the corresponding nonlabeled RNA. All cell experiments were performed in three biological replicates.