Fig. 3

AF10 prevents reprograming through its interaction with DOT1L. a Domain organization of wild type and mutant AF10s used in d. L107A mutation abolishes Histone H3K27 binding and OM-LZ deletion impairs DOT1L binding. b Immunoblots for AF10 following streptavidin pulldown from cells expressing BioID-DOT1L and GFP, WT-AF10 or OM-LZ∆ AF10. Top panels show 2% of input samples and bottom panels show Streptavidin pull-down samples. (+) Biotin cells were treated with 50 μM D-Biotin 24 h. c Immunoblots for AF10 following immunoprecipitation with IgG or HA antibodies form cells expressing DOT1L-HA and GFP, WT-AF10 or OM-LZ∆ AF10. Left panels show 2% of input samples and right panel shows HA pull-down samples. d Fold change in the number of Tra-1-60-positive colonies derived from control or AF10 knock-out cells expressing WT, L107A or OM-LZ∆ AF10 cDNAs. P values were determined by one sample t test; *P < 0.05. Bar graph shows the mean and error bars represent SEM in 3 independent biological replicates. Bottom panel shows the H3K79me2 levels with H3 total as a loading control. e Relative cell viability of dH1f cells expressing WT, L107A or OM-LZ∆ AF10. Cell Titer Glo Assay measurements were normalized to uninfected dH1f cells across the indicated time-points. Error bars indicate standard deviation of triplicate samples