Trodusquemine Inhibits Amyloid Oligomer Binding

SH-SY5Y cells were seeded on glass coverslips and treated for 15 min with HypF-N oligomers (6 µM) or Aβ₄₀ oligomers (5 µM) or in the absence or presence of increasing concentrations of trodusquemine (10:1, 3:1, and 1:1 ratios of oligomers to trodusquemine, monomer equivalents). After incubation, the cells were washed with PBS and counterstained with 5.0 µg mL⁻¹ Alexa Fluor 633-conjugated wheat germ agglutinin (Life Technologies, CA, USA)^{22 (link),28 (link)}. After washing with PBS, the presence of Aβ₄₀ or HypF-N oligomers was detected with 1:800 diluted mouse monoclonal 6E10 anti-Aβ antibodies (BioLegend, CA, USA) or 1:800 diluted rabbit monoclonal anti-HypF-N antibodies (Primm, Milan, Italy) and subsequently with 1:1000 diluted Alexa Fluor 488-conjugated anti-mouse or anti-rabbit secondary antibodies (Life Technologies, CA, USA) to recognize the 6E10 and anti-HypF-N primary antibodies, respectively. Fluorescence emission was detected after double excitation at 488 nm and 633 nm by the scanning confocal microscopy system described previously^{22 (link)} and three apical sections were projected as a single composite image by superimposition. The percentages of oligomer colocalization were determined by analyzing regions of interest corresponding to 50–60 cells per condition.