Higher frequency of derepression of Nanog and Sox2 genes in 4F2A if-iPSC colonies than NGFP if-iPSC colonies. (a)–(c) Expression levels of Nanog to Oct4 genes in NGF if-iPSC, 4F2A if-iPSC, and mESC colonies. Expressions of Nanog (upper band) and Oct4 (lower) in individual if-iPSC colonies are presented in gel images (a) and by statistics using a box-scatter plot (b). Oct4 expression levels are shown in (c). In (a) and (b) the expression levels of Nanog and Oct4 are of their own, not normalized with Gapdh. The values of individual colonies are shown as dots on the left of each box plot. The mean values are indicated in the boxes (circles). Box whiskers indicate the range from 5% to 95%. (d) Duplex RT-PCR showing expression levels of Sox2 relative to Gapdh in single colonies. Expression frequency is higher in 4F2A if-iPSC colonies than in NGFP if-iPSC colonies. The primer sets used were designed to specifically recognize 3′-untranslated regions (UTR) of endogenous Oct4, Nanog, and Sox2 genes which were absent in NGFP and 4F2A reprogramming cassettes. Gapdh, internal control.