Figure 11. AIM-100-induced DAT endocytosis is independent on dynamin-2, actin cytoskeleton, cdc42 and cholesterol-rich lipid rafts.

(A) PAE/YFP-HA-DAT cells were transfected twice with non-targeting (NT) or dynamin-2 (Dyn2) siRNAs. After 3–5 days, the cells were lysed and tested for the efficiency of knock-downs or for microscopy imaging. Cells were incubated with HA11 for 45 min at 37°C, and then incubated with (vehicle) or 20 µM AIM-100 for 2 hr at 37°C. 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), 561 (Cy3) and 640 nm (Cy5) channels. Cy3/Cy5 ratios were calculated, and the results are presented as mean values of the ratio (±SD, n = 5-6). P values are for ‘AIM-100’ compared to ‘vehicle’. (B) Cells were incubated with HA11 for 1 hr at 37°C, and then incubated with vehicles corresponding to ML141 (10 µM), FIPI (1 µM), Latranculin B (LatB, 0.4 µM) or nystatin (25 µM) for 15 min at 37°C. Cells were then incubated with (DMSO; vehicle-2) or 20 µM AIM-100 for 2 hr at 37°C in the presence of inhibitors. After fixation, cultures were stained and imaged as in (A). Cy3/Cy5 ratios were calculated, and the results are presented as mean values of the ratio (±SD, n = 6) relative to this value in vehicle-pretreated/AIM-100-treated cells (grey bar). P values are for ‘AIM-100 plus an inhibitor’ versus ‘AIM-100 plus vehicle’, unless indicated otherwise on the graph.