Inhibition of ADAM10 affects Notch targeted Hes5 and Gli genes in the ventral spinal cord (A–H) and in vitro cultured cell system (I–M).

(A–H) After electroporation at E4, transverse section at E6 was used for in situ hybridization. The ADAM10-mo transfected cells are green and the left side serves as a control. In situ hybridization using the antisense probes for Hes5 (B), Hes1 (C), Gli1 (F), Gli2 (G), and Gli3 (H) are labeled purple and cell nuclei are blue. Arrows indicate the downregulation of mRNA expression (purple color) in the transfected region (green). (I) Western blot analysis reveals that premature ADAM10 (pADAM10), the cleaved Notch1 (NICD), and Hes5 proteins are decreased in the ADAM10 siRNA transfected human neural progenitor cells at differentiation day 3, when compared to the control group (n = 2). GAPDH was used as a loading control. (J–L) Three days after differentiation, cells were stained against βIII-tubulin using antibody Tuj1 (green) in control siRNA (J) and ADAM10 siRNA transfected cells (K). Cell nuclei are labeled with DAPI (blue). Quantitative analyses reveal that the number of Tuj1-positive cells (L) significantly increase in the ADAM10 siRNA transfected cells compared to the control group (*p<0.05). Abbreviations: cle Notch1, cleaved Notch1. Scale bars, 200 µm in (A) for (A–H); 100 µm in (J) for (J, K).