FIGURE 6.

Mint2 overexpression inhibits NGF-induced neurite outgrowth in cultured DRG neurons. A, Mint2 RNAi efficiency was assessed by Western blot analysis. HEK293 cells were transfected with Myc-Mint2 (lane 1), Myc-Mint2 and nonsense-RNAi (lane 2), Myc-Mint2 and the Mint2-siRNA constructs at 0.5 μg (lane 3, No.1; lane 5, No.2; lane 7, No.3) or 1 μg (lane 4, No.1; lane 6, No.2; lane 8, No.3). Mint2 was detected by blotting with a monoclonal anti-Myc antibody. Actin was used as a loading control. B, GFP-tagged Mint2-siRNA or GFP-tagged nonsense-siRNA plasmids were transfected into primary rat DRG neuron cultures (left panels). 24 h post-transfection cultures were immunostained with anti-Mint2 antibody to examine the expression of Mint2 (right panels). Scale bar, 10 μm. C, primary rat DRG neuron cultures were transfected with GFP tagged nonsense-siRNA, Mint2/GFP, or Mint2-siRNA plasmids and subsequently incubated with NGF for 3 days. The transfected neurons were visualized as both GFP (green) positive and MAP2 staining (red) positive. Scale bar, 20 μm. D and E, quantification of neurite outgrowth in cultured DRG neurons. Image-pro Plus analysis software was used to quantify the length of the longest neurite (D), and Neurolucida software analysis software was used to quantify total neurite length (E). Data are presented as the means ± S.D. The double asterisk indicates significant difference (p < 0.01, Student's t test) compared with control.