Fig. 4

Adenoviral-mediated overexpression of 3F-SMN protein in HepG2 cells leads to the release of EVs containing high levels of the 3F-SMN protein. Panel A: HepG2 cells were infected with Ad3F-SMN at an MOI = 50, 100, 200, or mock infected (PBS). Seventy-two hpi, cell lysates were collected and EVs were isolated using polyethylene glycol (PEG). Protein content of the EVs was examined by immunoblot. Endogenous SMN protein is indicated by a grey arrow and 3F-SMN protein is indicated by a black arrow. The red square indicates the region used for quantification of SMN protein signal for Panel B and C. Panel B: SMN protein signal in cell lysates were quantified, and the fold change in SMN protein signal relative to endogenous SMN protein signal is shown. Panel C: SMN protein signal in EVs was quantified and presented as the fold change in SMN protein signal relative to endogenous SMN protein signal. Panel D: Particle size and diameter of isolated EVs was assessed by nanoparticle tracking analysis. In panels B and C, significance was calculated after transforming data by log transformation, then tested for significance using one-way ANOVA followed by Tukey’s multiple comparisons test. An asterisk (*) indicates p < 0.05, two asterisk (**) indicates p < 0.01, and three asterisk (***) indicates p < 0.001. Data represents 3 independent experiments.