Figure 3. STRAP regulates the expression of CSC markers and NOTCH signaling genes.

A. Western blot was used to determine the expression of protein levels for indicated CSC markers in control and STRAP knockdown clones. B. Immunofluorescence staining of SOX2- and NANOG-positive cells in representative clones (Green). Cell nuclei were stained using DAPI stain (blue). C. Comparison of the relative expression (intensity) of SOX2 and NANOG from above staining in indicated KD cells and their control cells has been shown as box plots. D. Levels of mRNA in stable shSTRAP#1, shSTRAP#2, and shCtrl clones (HCT116 and DLD-1) as indicated were determined by qPCR and shown as the relative fold changes using GAPDH as a loading control. Significance levels were determined by Student’s t test. n=3, *P<0.05, when compared with the control. E. Protein expression of NOTCH components in shSTRAP#1 and shCtrl from HCT116 and DLD-1 cell lines was analyzed by western blot using antibodies against the corresponding proteins as indicated. F. Expression of HES1 was rescued by Flag-tagged STRAP adenovirus in shSTRAP#1 clone in a dose-dependent manner with β-gal adenovirus as a negative control. Endogenous and exogenous protein levels of STRAP were detected. G. Luciferase activities from HES1 and HES5 promoter reporters in STRAP knockdown and control cells normalized to β-gal activities were plotted. n=3, *P<0.05, when compared to the control.