Fig 1. Ubtor depletion promotes neurite outgrowth in hippocampal neurons and ld-PC12 cells.

(A) Neurite outgrowth in primary culture of rat hippocampal neurons. Dissociated hippocampal neurons were transfected with either Cy3-labeled negative control siRNA (NC) or Ubtor siRNA (Ubtor siKD) and then cultured in vitro for 36 hrs (HIV). Neurites were stained by the acetylated tubulin antibody. Transfected cells were indicated by the Cy3 fluorescence signals from the Cy3-labeled siRNAs. Scale bar, 50 μm. (B) Quantitative analysis of neurite outgrowth at 36 HIV. Neurite lengths were measured from 15 images for the NC, and 16 images for the Ubtor siKD groups, taken from 3 independent experiments. n = 241 and 288 for the NC and the Ubtor siKD group, respectively. t = 12.71, df = 527, P < 0.0001. (C) Immunoblot analysis of siRNA mediated knock down in hippocampal neurons. t = 8.427, df = 2, P = 0.0138. (D) NGF-induced neurite outgrowths in ld-PC12 cells transfected with either Cy3-labeled negative control siRNA (NC) or Ubtor siRNA (Ubtor siKD). Transfected cells were serum-starved overnight and treated with 50 ng/ml of NGF for 0 and 48 hours. Scale bar, 50 μm. (E) Quantitative analysis of neurite outgrowth at 48 hours post NGF treatment. Neurite outgrowth rates were calculated from 38 images for the NC, and 36 images for the Ubtor siKD groups, taken from four independent experiments. t = 9.827, df = 72, P < 0.0001. Neurite lengths of differentiated cells were measured in these images. n = 88 and 243 for the NC and the Ubtor siKD group, respectively. t = 7.721, df = 329, P < 0.0001. (F) qRT-PCR analysis of Ubtor expression levels. Expression levels relative to GAPDH levels are normalized to the untreated NC group. Three biological repeats, F_{(2, 6)} = 181.1, P < 0.0001. Multiple comparison significance values are indicated on the graph. (G) Immunoblot analysis of siRNA mediated knock down in ld-PC12 cells. t = 14.94, df = 2, P < 0.01.