Figure 3.
TSC deficiency induces multiple axons in culture. (A–D) Knockdown of Tsc2 by shRNA or knockout of Tsc1 induced multiple axons in vitro. (A) Tsc2 protein level in E18 rat hippocampal neurons infected with a lentivirus expressing control shRNA or shRNA against Tsc2 (shTsc2). (B) E18 rat hippocampal neurons transfected with either EGFP alone or EGFP together with Tsc2 shRNA. Tsc2 knockdown induced multiple axons, all of which are positive for Tau1. (*) P < 0.001 for multiple axons between the two groups by ANOVA. (C) Tsc1 protein level in E17 Tsc1flox/flox mouse hippocampal neurons infected with a control or Cre-expressing lentivirus. Neurons were lysed for Western blot analysis at 6DIV. (D) E17 mouse hippocampal neurons from Tsc1flox/flox embryos transfected with EGFP alone or EGFP together with Cre. Arrowheads indicate axons positive for Tau1. (*) P < 0.001 for multiple axons between the two groups by ANOVA. The data represent the mean ± SD of 135–185 neurons per condition from three independent experiments. Bars, 20 μm.