Figure 6. Dopamine, amphetamine and modafinil inhibit AIM-100-induced oligomerization and endocytosis of DAT.

(A) PAE/YFP-HA-DAT cells were incubated with HA11 for 30 min at 37°C, and then incubated with vehicle (water), 100 µM dopamine (DA), 100 µM amphetamine (Amph) or 20 µM modafinil (Mod) for 15 min. The cells were further incubated with vehicle (DMSO) or 20 µM AIM-100 for 2 hr at 37°C in the same media. After fixation, cultures were stained with secondary anti-mouse antibodies conjugated with Cy5 (surface HA-DAT), permeabilized with Triton X-100 and stained with secondary anti-mouse conjugated with Cy3 (internalized HA-DAT). 3D images were acquired through 488 (YFP, not shown), 561 (Cy3, red) and 640 nm (Cy5, cyan) channels. Maximum intensity projection images are presented. Scale bar. 10 µm. (B) Cy3/Cy5 ratios were calculated in experiments exemplified in (B). Results are presented as mean values of the ratio (±S.D.; n = 4–6). P values are calculated for ‘AIM-100’ against other experimental variants treated with AIM-100 and inhibitors/substrates. (C) PAE/YFP-HA-DAT cells were incubated with vehicle, 100 µM DA, 100 µM Amph, 20 µM Mod or 10 µM cocaine (Coc) for 10 min. The cells were further incubated with DMSO or 20 µM AIM-100 for 2 hr at 37°C in the same media, and lysates were analyzed by immunoblotting with the GFP antibody. Representative experiment is shown. M, monomers; O, oligomers. (D) The mean values of the O/M ratio (±S.D.) were calculated in three independent experiments exemplified in (C), and expressed as percent of the O/M value determined in cells incubated with AIM-100 but not with inhibitors or substrates (‘veh-AIM-100’). P values are calculated for cells treated with AIM-100 and inhibitors/substrates versus ‘veh-AIM-100’.