Fig. 2

AF10 regulates H3K79 methylation and is a barrier to reprogramming. a Schematic for AF10 (MLLT10) gene indicating target sites for the AF10 sgRNAs. b H3K79me2 upon sgRNA-mediated AF10 knock-out. iDOT1L (EPZ004777) was used as a positive control of H3K79me2 depletion. Fibroblasts were treated with DMSO or 3 μM iDOT1L for 10 days. sgAF10 infected dH1fs were selected with puromycin and cultured for 1 week. Total H3 levels are used as loading control. Quantifications were normalized to sgControl sample. c Fold change in the number of Tra-1-60 positive colonies upon sgAF10 expression. P values were determined by one sample t-test; *P < 0.05. Bar graphs show the mean and error bars represent SEM in independent biological replicates (each circle). Representative Tra-1-60 stained wells are shown below the graph. P values were 0.009 for sgAF10-1 and 0.016 for sgAF10-2. d Immunoblot for H3K79me2 in single-cell clones of sgControl and sgAF10 iPSC lines. Total H3 levels were used as loading control. e OCT4, SSEA4 and NANOG immunofluorescence of iPSCs derived from sgControl and sgAF10 expressing fibroblasts. DAPI was used to stain the nuclei. Scale bars represent 50 μm. f Hematoxylin and eosin stained sections of teratomas generated by iPSCs derived from sgControl and sgAF10-1 cells. Black arrowheads show glandular epithelium (endoderm, top), cartilage tissue (mesoderm, middle), and pigmented neural tissue (ectoderm, bottom). Representative images are from one of two independent teratomas. Scale bars represent 20 μm