FIG. 2.

Caspr4 inhibits the proliferation while promoting neuronal differentiation of NPCs. (A) NPCs were transfected with Caspr4 small interference RNA (siRNA) and control siRNA (NC), respectively. Transfected cells were stained for 5-bromo-2-deoxyuridine (BrdU) and 4′,6-diamidino-2-phenylindole (DAPI) after being cultured in the medium containing BrdU for 3 h. The numbers of BrdU⁺ cells were counted and expressed as the percentage of the number of DAPI⁺ cells (NC: 26.89±1.26; Caspr4-siRNA: 35.11±2.06). (B) NPCs were transfected with Caspr4 plasmid in a pCDF1-MCS1-EF1-copGFP vector that expresses coGFP (Caspr4) or in an empty vector containing only copGFP (GFP). After 3–4 days of differentiation in vitro, cells were immunostained for βIII tubulin (TUJ1), GFP, and DAPI. The numbers of GFP⁺TUJ1⁺ cells were counted and expressed as the percentage of the number of GFP⁺ cells (GFP: 15.33±1.01; Caspr4: 24.33±1.70). (C) NPCs were transfected with Caspr4 small hairpin RNA (shRNA) or Caspr4 shRNA plus intracellular domain of Caspr4 (C4ICD) as well as a scrambled shRNA (NC) as the control. After 3–4 days of differentiation in vitro, cells were immunostained for βIII tubulin (TUJ1), GFP, and DAPI. The numbers of GFP⁺TUJ1⁺ cells were counted and expressed as the percentage of the number of GFP⁺ cells (NC: 18.59±1.97; Caspr4-shRNA: 7.69±0.80; Caspr4-shRNA plus C4ICD: 14.89±1.82). Scale bars: 50 μm (A–C); 20 μm (B). Values are represented as mean±standard error of the mean (SEM), Student's t-test (A, B); one-way analysis of variance (C). **P<0.01. ***P<0.001. NS, not significant. Color images available online at www.liebertpub.com/scd