Perturbing the Normal Level of SIDT1 Suppresses the Naked ASO Effect

Figure 3

Microscopic analysis for the cellular uptake of naked Alexa568-labeled A-15. (a) MEFs were transfected with the empty plasmid (control) or pCI-neo-SIDT1. After the 48-hour culture, the cells were rinsed with PBS and further cultured in the absence (–) or presence (+) of 500 nM of naked A-15 for 24 hours. After washing the cells with PBS, they were analyzed by confocal microscopy. The Alexa568 fluorescence intensity in the SIDT1-overexpressing cells relative to that in the control cells is shown. Error bars indicate s.d. (). (b) MEFs were transfected with siControl, siSIDT1#1, or siSIDT1#2. After the 72-hour culture, the cells were rinsed with PBS and further cultured in the absence (–) or presence (+) of 500 nM of naked A-15 for 6 hours. After washing the cells with PBS, they were analyzed by confocal microscopy. The Alexa568 fluorescence intensity in the SIDT1 knockdown cells relative to that in the control cells is shown. Error bars indicate s.d. (). (c) MEFs were transfected with the empty plasmid (control) or pCI-neo-SIDT1. After the 48-hour culture, the cells were rinsed with PBS and further cultured in the presence of 500 nM of naked Alexa568-labeled A-15 for 24 hours. After washing the cells with PBS, fluorescence images from Alexa568 and LysoTracker Green were visualized using a confocal microscope. Colocalization rates were quantified with ImageJ. Error bars indicate s.d. ().

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