Figure 2. In cultured medium spiny neurons (MSNs), Mtss1 expression is directly regulated by Sema3E-Plexin-D1 signaling through the AKT pathway.

(A) Western blot images showing Mtss1 expression in MSNs derived from the striatum of wild-type (WT) or Plxnd1 conditional knockout (cKO) mice at P0 and measured at DIV3 and DIV6 in culture. (B) Quantification of band intensity in (A). Two-way ANOVA with Tukey’s post hoc correction for multiple comparisons; n = 3. (C) Mtss1 expression in MSNs obtained from the striatum of WT or Sema3e KO mice at P0 and measured at DIV6 in culture. (D) Quantification of the blots shown in (C). Student’s t-test; n = 5 for WT, n = 5 for KO in five independent experiments. (E) Schematic illustration of the experimental strategy for Sema3E-ligand or MK2206, an AKT inhibitor treatment in MSN culture. (F, G) Western blot images showing Mtss1 expression after AP-Sema3E (2 nM) treatment in cultured MSNs derived from Sema3e KO mice or Plxnd1 cKO mice. (H, I) Quantification of (F, G). Student’s t-test; AP, n = 4, AP-sema3E, n = 4 for sema3e KO mice, AP n = 4, AP-sema3E n = 4 for Plxnd1 cKO mice in three independent experiments. (J, K) Western blot to analyze the expression of Mtss1 and Plexin-D1 after MK2206 (100 nM) treatment in cultured MSNs and subsequent quantification for band intensity (L, M). Student’s t-test; n = 6 for sham, n = 6 for MK2206 in six independent experiments. (N O) Western blot image and analysis showing Mtss1 expression in Sema3e knockout MSNs treated with MK2206 after incubation with AP-Sema3E. Two-way ANOVA with Tukey’s post hoc correction for multiple comparisons; n = 5 in five independent experiments. Error bars, mean ± SEM; *p<0.05, **p<0.01, ***p<0.001 by indicated statistical tests. .