Uptake pathways of mRNA polyplexes by HeLa Cells. (A) Red fluorescent labeled V5PB mRNA compacted to PEI were administered to HeLa cells. Clathrin detection was performed by immunofluorescence using an anti-clathrin primary antibody revealed with an Alexa-488 conjugated secondary antibody (green). Experiments have been done three times. (B and C) Red fluorescent-labeled V5PB mRNA was used to transfect HeLa cells constitutively expressing green fluorescent caveolin-1 (B) or green fluorescent Rab7 proteins (C). Experiments have been done three times. For all experiments, the route of the labeled polyplexes was observed at 30 min and 2 h post-transfection using confocal microscopy. Yellow dots correspond to colocalization of red and green fluorescent spots. Insets shows enlargement of boxed areas with colors separated. BF: Bright Field. Nuclei were stained in blue by lamin immunodetection (Caveolin and Rab7 experiments) or DRAQ5® (Clathrin experiment). The images shown are representative Z-sections from three separated experiments.