Figure 4. Suppression of Notch activation prevents Smad7 expression. A: P0 limbal epithelial stem cells (LSCs) were treated with 10 μM of 4-(1,3,3a,4,7,7a-Hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl)-N-8-quinolinyl-Benzamide
(IWR-1), cyclopamine, or N-(N-[3,5-difluorophenacetyl]-l-alanyl)-S-phenylglycine t-butyl ester (DAPT) for 48 h. Cell extracts
were then harvested for western blotting. A representative picture of three independent experiments is shown. B: DAPT dose-dependently suppresses Smad7 expression. Representative blots (below panels) and densitometric analysis with standard
deviation (SD; upper columns) of three independent experiments are shown. *p<0.001 versus untreated cells. C: Hes1 and Smad7 mRNA in P0 LSCs assayed with quantitative real-time PCR (qPCR). The cycle threshold (Ct) value of the Hes1 or Smad7 PCR product and control mRNA (GAPDH) were used to calculate the relative quantities of mRNA between
the samples. Data represent three independent experiments. *p<0.005 versus untreated cells. **p<0.002 versus untreated cells.