Figure 7. Notch activation involves in the upregulation of Smad7 expression and blocks transforming growth factor-beta signaling in limbal epithelial stem cells. A: Western blot analysis of Jagged-1 effect on the induction of the Notch intracellular domain (NICD) and Smad7 in P1 limbal epithelial stem cells (LSCs). P1 cells were treated with different doses of Jagged-1 (1 and 10 μM) for 12 h and 24 h to induce the NICD (approximately 100 kDa) and Smad7/Hes1, respectively. P1 cells were also pretreated with N-(N-[3,5-difluorophenacetyl]-l-alanyl)-S-phenylglycine t-butyl ester (DAPT) for 1 h before jagged-1 treatment for additional 12 h and 24 h. Immunoblot results are from a representative experiment performed in triplicate with β-actin as loading control. B: quantitative real-time PCR (qPCR) analysis of the Jagged-1 effect on transforming growth factor-beta (TGFβ)-mediated growth suppression. Cells were pretreated with 1 μM Jagged-1 for 24 h and then treated with TGFβ1 for further 24 h. Subsequently, total RNA was extracted for determining Ki67 mRNA with the qPCR assay as described in Figure 2C. Data represent three independent experiments. C: Western blot analysis of the Jagged-1 effect on TGFβ-mediated induction of mesenchymal markers. Cells were pretreated with 1 μM Jagged-1 for 24 h and then treated with TGFβ1 for a further 24 h. Representative blots (left panels) and densitometric analysis with standard deviation (SD; right columns) of three independent experiments are shown.