Fig. 1: Detecting cytosolic delivery of siRNA during sub-nanomolar siRNA transfection.

Airyscan confocal imaging of HeLa cells during treatment with lipoplexed AF647-siRNA. a HeLa cells stably expressing d1-eGFP exhibiting the cytosolic distribution of AF647-siRNA after 4 h siRNA treatment. The scale bar is 50 µm. Representative images of eight independent experiments. b HeLa cells expressing YFP-galectin-9 were imaged every 5 s during siRNA transfection, to visualize de novo galectin-9 recruitment to lipoplex-containing vesicles, followed by endosomal release and gradual cytosolic dispersion of AF647-siRNA. Outlines indicate cell boundaries and arrows indicate the releasing particle. The scale bar is 20 µm. Representative images of three independent experiments. c Representative images showing redistribution of AF647-siRNA to cytoplasmic foci after endosomal release and initial homogenous cytosolic dispersion. The scale bar is 20 µm. Images are representative of eight independent experiments. d The redistribution of siRNA-AF647(as) into cytoplasmic foci after release to the cytosol was monitored in cells expressing GFP-AGO2. The image detail shows a single AGO2⁺ structure. Scale bars, 20 µm; details, 2 µm. Representative images of two experiments. Cells were incubated with 0.4 nM (a, c) or 0.67 nM (b, d) siRNA.