Figure 1.

Optimization of the read-out for the siRNA screen. (A) Representative images of GFP-positive HBG3 ES cell-derived motor neurons (green) reverse transfected with Vamp2 siRNA using Dreamfect Gold in a 96-well plate format and immunostained for VAMP2 (red). Scale bar, 20 μm. (B) Western blot analysis of VAMP2 after siRNA mediated Vamp2 knock-down in HBG3 ES cell-derived motor neurons transfected in a 96-well format. (C) HBG3 ES cell-derived motor neurons expressing GFP (upper panels) were incubated with AlexaFluor555-conjugated H_CT (middle panels) and αp75^NTR (lower panels) in the presence of 1 mM EHNA or DMSO vehicle control for 2 h at 37°C. Cells were then acid-washed, fixed and immunostained with AlexaFluor488-conjugated anti-rabbit IgG. The intracellular accumulation of both probes was substantially reduced by EHNA treatment, indicating that cytoplasmic dynein plays a major role in this process. Scale bar, 20 μm. (D,E) Quantification of internalized H_CT (D) and αp75^NTR (E) in motor neurons exposed to H_CT and αp75^NTR from three independent experiments with or without EHNA (paired t-test, mean ± s.e.m., ^***p = 0.001).