FIG. 6.

LNX2 is a downstream element of Caspr4 in promoting neuronal differentiation of NPCs. (A) Caspr4 were cotransfected with LNX2 or the empty vector into HEK293T cells. The cells were harvested at 48 h after transfection and subjected to western blot analysis using antibodies against Caspr4 and LNX2. γ-Tubulin (tubulin) was detected as a loading control. Quantification analysis of the blots was shown. (B) NPCs were transfected with LNX2 siRNA or a scrambled siRNA (NC) as a control. The cells were harvested at 48 h after transfection and subjected to western blot analysis using antibodies against Caspr4, LNX2, and Sox2. γ-Tubulin (tubulin) was detected as a loading control. Quantification analysis of the blots was shown. (C) LNX2 were cotransfected with Caspr4 or the empty vector into HEK293T cells. The cells were harvested at 48 h after transfection and subjected to western blot analysis using antibodies against Caspr4 and LNX2. γ-Tubulin (tubulin) was detected as a loading control. Quantification analysis of the blots was shown. (D) NPCs were transfected with Caspr4 shRNA, Caspr4 shRNA plus LNX2, or a scrambled shRNA (NC) as a control. Cells were immunostained for βIII tubulin (TUJ1), GFP, and DAPI after 3–4 days of differentiation in vitro. The numbers of GFP⁺TUJ1⁺ neurons were counted and expressed as the percentage of the number of total GFP⁺ cells (NC: 29.23±1.37; Caspr4-shRNA:15.46±0.78; Caspr4-shRNA plus LNX2: 28.20±1.48). (E) NPCs transfected with LNX2 shRNA or LNX2 shRNA plus C4ICD or a scrambled shRNA (NC) were immunostained for βIII tubulin (TUJ1), GFP, and DAPI after 3–4 days of differentiation in vitro. The numbers of GFP⁺TUJ1⁺ neurons were counted and expressed as the percentage of the number of total GFP⁺ cells (NC: 14.84±1.79; LNX2 shRNA: 7.24±1.31; LNX2 shRNA plus C4ICD: 5.04±0.85). Scale bars: 50 μm. Values are represented as mean±SEM. *P<0.05; **P<0.01; ***P<0.001; NS, non-significant. Color images available online at www.liebertpub.com/scd