Fig. 2

Morphological and molecular characterization of induced keratinocytes. a Induced keratinocytes (iKCs) exhibit keratinocyte-like morphological properties, closely resembling primary keratinocytes (1°KCs) in culture. iKCs displayed a round shape and formed colonies with a cobblestone-like appearance, a characteristic of primary keratinocytes in culture. In contrast, parental mouse embryonic fibroblasts (MEFs) exhibit mesenchymal characteristics, as they appear spindle shaped and do not form colonies. Scale bars = 25 μm. (b) Expression levels of keratinocyte and fibroblast markers were measured by semi-quantitative RT-PCR, and agarose gel electrophoresis showing the product of each PCR reaction is depicted. The relative levels of each product from three independent experiments were normalized to actin. Results are depicted as fold-change of expression compared to expression in either primary keratinocytes (K14, K5, and p63) or in MEFs (vimentin). Error bars represent standard error between experiments. In contrast to MEFs, iKCs expressed undifferentiated keratinocyte markers (K14 and K5) at levels comparable to those of primary keratinocytes; p63 expression was threefold higher in iKCs compared to MEFs, even though KCs expressed p63 at much higher levels; vimentin expression was reduced twofold in iKCs, albeit not completely abolished. c Immunofluorescence staining against vimentin (Vim) and keratin 14 (K14) in MEFs, primary KCs, and iKCs. DAPI staining is shown in blue, and Vim or K14 staining is shown in green. Scale bars = 20 μm. (d) Western blot analysis to measure vimentin and K14 protein levels in MEFs and iKCs. Total cellular protein lysates were run and blotted with anti-vimentin and anti-K14 antibodies, as well as with anti-actin as a control. e Endogenous and total expression levels of pluripotency factors Klf4, Sox2, and Oct4 were assessed by RT-PCR in MEFs, KCs, and iKCs. ESCs were included as a positive control. Expression of these factors is silenced in iKCs