Figure 3 | Scientific Reports

Figure 3

From: Endocytic uptake of monomeric amyloid-β peptides is clathrin- and dynamin-independent and results in selective accumulation of Aβ(1–42) compared to Aβ(1–40)

Figure 3

Uptake of Aβ(1–40), Aβ(1–42) and Trf in SH-SY5Y cells under conditions that perturb dynamin dependent endocytosis. (a) Uptake of HF488-labelled Aβ(1–40), Aβ(1–42) and AF647-labelled Trf in cells treated with dynasore (80 µM). The peptide uptake is reported as mean cellular uptake relative to control (cells not treated with inhibitor) for 4 independent experiments, each performed in triplicate. (b) Correlation analysis of the data presented in (a) with R2 of the linear fit of average inhibitions levels from each experiment and treatment, and Pearson’s correlation coefficient (r). (c) Statistical analysis of the data in (a) performed by one-way ANOVA with matched data followed by multiple comparisons with Bonferroni post-hoc test. The table shows the adjusted p-values for the individual comparisons made (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). (d) Mean cellular intensity ± SD (N = 2, n = 3–5) of live cells after incubation with HF647-labelled Aβ(1–40) or Aβ(1–42). (e) Flow cytometry histogram of live cells 24 h post transfection with EGFP-labelled dyn2 K44A: the cells were gated for peptide uptake based on transfection efficiency and the extent of dyn K44A overexpression as measured by the intensity of the EGFP label. (f) Uptake of HF647-labelled Aβ(1–40), Aβ(1–42) or AF647-labelled Trf in dyn2 K44A transfected cells, gated as in (e) and related to peptide uptake in cells gated as non-transfected (N = 2, n = 3–5). (g–i) Confocal imaging of dyn2 K44A (green) transfected cells imaged 24 h post transfection and incubated with HF647-labelled (g) Aβ(1–40), (h) Aβ(1–42) or (i) AF647-labelled Trf (red). In all experiments the concentration of Aβ was 1 μM and the concentration of Trf was 5 µg/ml. Cells were incubated with Aβ for 1 h and Trf for 5 min (flow cytometry) or 10 min (confocal microscopy). Relative uptake was calculated based on mean cellular fluorescence intensity ± SD of the total number of gated live cells measured by flow cytometry. All flow cytometry data were corrected for baseline contributions by subtracting the cellular autofluorescence.

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