In vitro characteristics of the protein-iPS cells. (A) RT-PCR of the ES cells, cFB-protein-iPS cells, cFBs, and feeder cells (STO). RNA of cFB-protein-iPS cells was harvested from 4 clones. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control (right panels). The gene expressions of Oct4 and Nanog were verified by real-time PCR (left panels). There is no significant difference in the expression levels of Oct4 and Nanog between ES and protein-iPS cells. The expression levels in ES and protein-iPS cells are at least 10 000-fold higher than those in fibroblasts. (B) Scatterplots as determined by microarrays (n = 3 each). The global gene expression patterns were compared between the cFB and cFB-protein-iPS cell, and the ES cell and cFB-protein-iPS cell. The red lines indicate 2-fold changes in log scale. (C) Hierarchical clustering based on 4503 differentially expressed genes. (D) DNA methylation status of the Oct4 and Nanog promoters using bisulphite sequencing. ○ indicates unmethylated CpG nucleotides, and ● indicates methylated CpGs. (E) Histone modification status of the promoters. ChIP assays of trimethylated H3K4 and H3K27. Conventional PCR (top panel) and real-time PCR (bottom panel, n = 3, each). Expression level was adjusted by GAPDH (ES cell = 1.0 as an arbitrary unit).