Fig. 7. Effects of the K133/K137 ubiquitination site mutation of Oct4 on Oct4-induced somatic cell reprogramming.

a Knockdown or overexpression of Kap1 in Oct4-GFP MEFs. The expression levels of Kap1 and GAPDH in the control, Kap1-silenced, and -overexpressed Oct4-GFP MEFs were determined by western blotting. b The effects of Kap1 knockdown or overexpression on the OSKM-induced reprogramming of Oct4-GFP MEFs. Oct4-GFP cells were stained with AP staining on day 10. c The GFP-positive colonies were captured by confocal microscopy (left panel), and was counted from the images. Scale bar = 500 μm. Data are expressed as mean ± SD (n = 9). d The GFP-positive population was measured by flow cytometric analysis. e The effect of Kap1 overexpression on OSK-induced reprogramming of Oct4-GFP MEFs. iPSC colonies were visualized with AP staining, and GFP-positive colonies were photographed (left panel). The number of GFP⁺ colonies was quantified 10 days after OSK expression (right panel). Data are expressed as mean ± SD (n = 9). (Scale bar = 300 μm). f The percentage of GFP-positive cells was quantified by flow cytometry analysis on 21 days. g Western blot analysis of Oct4 protein levels in Oct4-GFP MEFs infected with either a retrovirus encoding Oct4^WT or Oct4^K133R/K137R. h Representative images of AP⁺ colonies (upper images) and Oct4-GFP⁺ colonies (lower images) in Oct4-GFP MEFs infected with OSKM or O^K133R/K137RSKM. The number of GFP⁺ colonies was counted on day 10. Scale bar = 300 μm. i The relative reprogramming efficiencies were determined by measuring the Oct4-GFP⁺ population using flow cytometry. j Representative images of AP⁺ colonies (upper images) and Oct4-GFP⁺ colonies (lower images) in Oct4-GFP MEFs infected with O^WTSK or O^K133R/K137RSK. The number of GFP⁺ colonies was counted on 25 days after the expression of O^WTSK or O^K133R/K137RSK. Scale bar = 500 μm. k The relative reprogramming efficiencies were determined by measuring the Oct4-GFP⁺ population using flow cytometry.