FIGURE 4.

Class IIa HDACs enhance reprogramming by accelerating the MET phase. A, measurement of proliferation at different times in fibroblasts reprogrammed as indicated. An empty vector was added to OKS as a control for HDAC7. A representative experiment with triplicate items counted in duplicate (after detaching cells with trypsin) is shown. D, day. B, representative phase contrast and immunofluorescence photographs of fibroblasts reprogrammed with OKS/OKSM and HDAC4 or -7 or empty vector. Scale bars = 50 μm. E-cad, E-cadherin; Ctrl, control. C and D, representative Western blot analyses for E-cadherin using lysates of fibroblasts reprogrammed with OKS/OKSM and the indicated expression vectors or shRNA constructs. Lysates were taken at day 6 in all cases. E, number of GFP+ colonies in mammary epithelial cells (MECs) reprogrammed with OKS and HDAC7 or empty vector. Colonies were counted at day 21. F, semiquantitative PCR showing integration of the exogenous HDAC7 transgene in the genome of selected iPSC clones produced as in E. Untransduced MECs and pMXs plasmids containing the corresponding cDNA were used as controls. G, Western blot analysis of fibroblasts infected with iHDAC7-Dox and treated with doxycycline (Dox). H, number of GFP+ colonies in fibroblasts reprogrammed with OKS/OKSM and iHDAC7-Dox. Doxycycline was administered at the indicated time points. Double asterisks indicate p value < 0.01.