Fig. 1.
The expression of stemness factor and stem cell marker genes and proteins in SKNAS iCSCs at day 149, day 175, day 200, day 592, and day 609. The SKNAS sphere culture has never been treated with epigenetic modifiers, whereas the SKNAS iCSCs were initially treated with 5AdC (2.5 μM) for 5 d and then cultured in the sphere-forming condition without 5AdC for the period indicated (day 149, day 175, day 200, day 592, and day 609). The expression of proteins of interest was examined by Western blot analysis, as previously described (25). Fold change in protein expression based on the densitometry analysis of the protein examined against β-actin was shown. The expression of genes indicated was examined in duplicate by TaqMan qPCR, using gene-specific TaqMan Gene Expression Assays (Applied Biosystems). (A and B) Expression levels of the genes examined were presented as fold change in the SKNAS sphere culture or iCSCs over the monolayer cells at day 149 and day 175. The expression of the proteins indicated was also examined in the SKNAS monolayer cells, non-drug-treated spheres, and iCSCs at day 149, day 175, and day 200. (C) Proteins of interest were examined in SKNAS monolayer cells, non-drug-treated spheres, and iCSCs at day 592. (D) Immunocytochemical assay was performed on cell block preparations of SKNAS monolayer cells, non-drug-treated spheres, and iCSCs at day 609 to investigate expression levels of MYC, MYCN, SOX2, CXCR4, and p75NTR (see SI Materials and Methods for description of procedures).