XN inhibits Notch1 signaling pathway.

<p>A. Protein levels of intracellular domain (NICD), HES-1, an immediate downstream target of Notch1 signaling, cyclinD1, and survivin were analyzed by western blot after XN-treatment. Equal loading was confirmed by GAPDH. B. Notch1 inhibition by shRNA (seq #1 and seq #2) induced apoptosis similar to XN treatment. Functional Caspase assay confirmed the induction of apoptosis in Notch1 knock-down cell lines. A non-specific, no target, scrambled sequence (NS) as control was used. GAPDH was used as gel loading control. C. Effect of active Notch1 overexpression in XN treatment. Huh-7 and Hep3B cells were transfected with Notch1plasmid (pcDNA-Myc-ICN1) and treated with or without XN. Percent of growth was measured by MTT assay (n = 3; p = 0.05 compared to vector with XN10). D. Western analysis of (pcDNA-Myc-ICN1) or empty vector transfected Huh-7 and Hep3B cells treated with or without XN for the expression of the exogenous Notch1 protein. Myc-tag antibody was used to determine the level of exogenous Notch expression.</p

Effect of XN on human hepatocellular carcinoma cellular proliferation.

<p>A. Commonly used four human hepatocellular carcinoma cancer cell lines were treated with XN at indicated doses for 4 days and cytotoxicity was measured using MTT assay (n = 3; p>0.05 at 5 μM and above concentrations for all cancer cell lines compared to control treatment. B. Cells were treated with XN up to 15 μM for 4 days and cell viability was measured by colony formation assay and the number of colonies were counted and showed in bar graph. (p>0.05 at 5 μM and above concentrations compared to control treatment. C. Effects of XN on Huh-7 and Hep3B cell proliferation in real-time. Cells were treated with indicated concentrations of XN and cell proliferation was monitored in real time with the continuous presence of XN. The cells were photographed and the cell confluence was calculated using IncuCyte 2011A software. The changes in cell confluence are used as a surrogate marker of cell proliferation. Statistically significant (p<0.05) growth suppressions were observed at or above 10 μM XN compared to control.</p