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. 2018 Mar 6;11:228–242. doi: 10.1016/j.omtn.2018.02.011

Figure 3.

Figure 3

Aptamers Were Delivered by CADY into Cultured SK-N-SH Cells and Inhibited α-syn Aggregation

(A) The CADY/Alexa594-labeled F5R1 complexes were formed at molar ratio of 20/1 and overlaid onto SK-N-SH cells for 4 hr at 37°C. Cells were then washed and the fluorescence was detected by confocal laser microscopy. Scale bars, 25 μm. (B and C) The CADY/F5R1 complexes were formed at different molar ratios and overlaid onto SK-N-SH cells for 4 hr at 37°C. Cells were then washed and trypsinized, and the fluorescent cells were counted by flow cytometry. Results are reported for 100,000 cells (n = 5). (D) SK-N-SH cells were transfected with EGFP-α-syn constructs or EGFP vector as control. After 6 hr, the aptamers of F5R1 and F5R2 labeled with Alexa594 complexed with CADY were used to treat these cells, respectively. The EGFP-α-syn co-localization with F5R1 or F5R2 was detected by confocal laser microscopy. Arrows showed the co-localization spot. Scale bar, 10 μm. (E) SK-N-SH cells were pre-treated with CADY/biotinylated-aptamer complexes (aptamer at 100 nM and the CADY/aptamer molar ratio at 40; random DNA sequence served as the control), and then transfected with pcDNA3.1-α-syn for α-syn overexpression. Streptavidin magnetic beads were incubated with protein lysate overnight at 4°C. Samples were resolved by using SDS-PAGE and α-syn was detected by western blotting. (F) Principle of bioluminescent-protein complementation assay based on G. princeps luciferase. (G) SK-N-SH cells with/without CADY/aptamer complex pre-treatment were co-transfected with constructs of α-syn-hGLucN and α-syn-hGLucC. After transfection for 24 hr, the luciferase activity from protein complementation was measured in an automated plate reader at 480 nm with substrate coelenterazine (20 μM). Data are presented as the mean ± SD (one-way ANOVA) ***p < 0.001 compared with control group (n = 6); ###p < 0.001 compared with α-syn-hGLuN/C group. (H) SK-N-SH cells with/without CADY/aptamer complex pre-treatment were transfected with constructs of α-syn-hGLucN and α-syn-hGLucC to show the expression of each protein. Immunoblots were probed with antibody against α-syn.