Figure 1

Cellular uptake of α−syn and tau fibrils into SDC transfectants. WT K562 cells and SDC transfectants were incubated with either of the FITC-labeled fibrils (α−syn or tau at a concentration of 5 µM monomer equivalent) or Trf (25 µg/ml) for 3 h at 37 °C. Cellular uptake of the fibrils and Trf was then analyzed with flow cytometry and microscopy. (a) Scanning electron microscope visualization of α−syn and tau fibrils, along with WT K562 cells and SDC transfectants treated with the fibrils at 10 min and 3 h of incubation. (b) Flow cytometry histograms showing intracellular fluorescence of WT K562 cells and SDC transfectants, following 3 h incubation with fluorescent α−syn or tau fibrils or Trf. (c) Detected fluorescence intensities were normalized to fibril (α−syn or tau) or Trf-treated WT K562 cells as standards. The bars represent mean ± SEM of five independent experiments. Statistical significance vs standards was assessed by analysis of variance (ANOVA). *p < 0.05 vs standards; **p < 0.01 vs standards. (d) CLSM visualization of fibril (α−syn, tau) and Trf uptake. (e–g) Colocalization of α−syn or tau fibrils and SDCs. SDC transfectants treated with either of the fluorescent fibrils (α−syn or tau) or Trf for 3 h were permeabilized and treated with the respective APC-labeled SDC antibody. Nuclei of cells were stained with DAPI and cellular uptake was then analyzed with CLSM. Representative images of three independent experiments are shown. Scale bar = 10 μm. (h) SDS-PAGE showing FITC-labeled α-syn and tau fibrils immunoprecipitated with either SDC3 or SDC4 antibodies from extracts of SDC3 and SDC4 transfectants. Lane 1: 0.5 ug of FITC- α-syn; Lane 2: immunoprecipitate of FITC-α-syn-treated stable SDC3 transfectants; Lane 3: immunoprecipitate of untreated SDC3 transfectants; Lane 4: immunoprecipitate of FITC-α-syn-treated SDC4 transfectants; Lane 5: immunoprecipitate of untreated SDC4 transfectants; Lane 6: Molecular weight (MW) marker; Lane 7: 0.5 µg of FITC-tau; Lane 8: immunoprecipitate of FITC-tau-treated SDC3 transfectants; Lane 9: immunoprecipitate of untreated SDC3 transfectants. Lane 10: immunoprecipitate of FITC-tau-treated SDC4 transfectants; Lane 11: immunoprecipitate of untreated SDC4 transfectants. Standard protein size markers are indicated on the left.