Colon organoid formation and cryptogenesis are stimulated by growth factors secreted from myofibroblasts
Fig 6
The cYH2CM does not stimulate canonical Wnt and Notch signaling pathways.
The ability of the YH2CM to stimulate the Wnt or Notch signaling pathway was tested in cell lines by its ability to stabilize the β-catenin protein (for Wnt pathway) or Notch Intra-Cellular Domain protein (NICD, for Notch pathway). (A) L-cells were incubated with partially purified Wnt3a (0.2% and 0.5% v/v) or cYH2CM (10% and 50% v/v) for 4 hours before Western blotting for β-catenin and β-tubulin. The normalized β-catenin intensities were plotted indicating that cYH2CM does not stabilize β-catenin. (B) Western blot detecting β-catenin using mouse anti-human/mouse β-catenin monoclonal antibody (Clone 14/ β-catenin, BD Transduction Laboratories #610153) to probe the L-cell cell lysate. (C) HCT116 cells were incubated either with 50% (v/v) of DMEM (control) or cYH2CM for 1h, 3h and 72h. For EDTA treatment, the cells were incubated with EDTA/PBS (10 mM) for 15 min. Western blotting for NICD and β-tubulin was conducted on the cell lysate with the normalized NICD intensities plotted. cYH2CM does not stimulate NICD protein in β-catenin. (D) Western blot detecting NICD using rabbit anti-mouse cleaved Notch 1 monoclonal antibody (Clone D3B8, Cell Signaling #4147) to probe the HCT116 cell lysate. Error bars: SEM, n = 3, 2-way ANOVA Tukey’s test.