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. 2022 Feb 12;27:1249–1259. doi: 10.1016/j.omtn.2022.02.006

Figure 2.

Figure 2

The retention and recycling of HG1-9 conjugates within cells

(A) The accumulation of HG1-9 (AF488 conjugated, 50 nM) and hTf (AF647 conjugated, 50 nM) within HeLa cells along with incubation time (5, 15, 30, 60, 90, and 120 min) at 37°C; the fluorescence ratio of cells after incubation for 5 min was 1. (B) Gel analysis of the integrity of endocytic HG1-9 within cells, HG1-9 (TAMRA conjugated, 50 nM), or ctr-sq (TAMRA conjugated, 50 nM, negative control) was incubated with HeLa cells for 3.5 h; then cells were incubated in fresh medium for 0.5 h, where 20 pmol of free DNA was the reference. (C) Representative confocal images of the binding and distribution of aptamer HG1-9 within cells. Top row, HG1-9 (AF488 conjugated, 50 nM) was incubated with Rab7A-mCherry transfected cells for 60 min; middle row, HG1-9 (AF488 conjugated, 50 nM) incubated with LAMP-1-mCherry transfected cells for 120 min; bottom row, HG1-9 (TAMRA conjugated, 50 nM) and DND26 co-incubated with cells for 120 min; scale, 10 μm. (D) Time-resolved spinning disk confocal images of HG1-9 (AF488 conjugated, 50 nM) in Rab11A-mCherry transfected HeLa cells after incubation at 37°C for 3 to 33 min, and the corresponding MCC, the fractions of HG1-9 overlapping Rab11A are shown in each image; scale, 2 μm. (E) Representative confocal images of the distribution of HG1-9 (AF647 conjugated, 50 nM) in TfR-EGFP and Rab11A-mCherry co-transfected HeLa cells after incubation for 120 min at 37°C; scale, 10 μm. (F) The amount change of dye conjugated hTf and HG1-9 within cells after being recycled and competing by unlabeled ligands. HeLa cells were pre-incubated with AF647 conjugated-hTf (50 nM) and AF488 conjugated-HG1-9 (50 nM) for 15 min, respectively; then the cells were incubated with unlabeled HG1-9 (500 nM) or unlabeled hTf (500 nM) for different times (0, 30, 60, and 90 min). The fluorescence of cells before incubation with unlabeled ligand was 100%. The experiment was conducted at least twice independently, and each sample has three replicates. t test, ∗∗∗p < 0.005; ns, not significant.