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. 2018 Jun 21;13(6):e0199412. doi: 10.1371/journal.pone.0199412

Fig 6. The cYH2CM does not stimulate canonical Wnt and Notch signaling pathways.

Fig 6

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.