Extended Data Fig. 2: H3K27me3 levels in naïve and primed hESC.
a Phase contrast microscopy images of primed and naïve hESC, untreated or treated with EZH2i over 5 days showing similar growth and morphology. Data shown represent 2 independent experiments. Scale bars 100 µm. b Immunofluorescence microscopy showing H3K27me3 staining of naïve and primed hESC + /- EZH2i treatment. Stainings were performed in parallel in the same antibody dilutions and images were acquired with the same gain settings to allow a quantitative comparison. Data shown represent 2 independent experiments. Scale bars 10 µm. c Representative western blot for quantification of H3K4me3, H2Aub and H3K27me3 assayed using two-color IR western blot, in hESC and WT(J1) mouse ESC (grown in 2i/Serum) with and without EZH2i treatment. d Quantification of western blots, represented as mean of two biological replicates. Representative image shown. We note that the discrepancy in fold-change comparing naïve and primed hESC with different quantitative methodologies MINUTE-ChIP, IF, western blots may arise from method-specific threshold sensitivity, dose-response curves and signal saturation levels. MINUTE-ChIP measures H3K27me3-density on the level of nucleosomes, whereas western blot yields a per-histone level quantification. We have previously confirmed that MINUTE-ChIP signal is linearly proportional under the dynamic range relevant to the H3K27me3 levels assayed42. e Time course of H3K27me3-depletion using immunofluorescence microscopy. Boxplot showing quantification performed using CellProfiler image analysis of 1172 nuclei (237 untreated, 140 day 2, 314 day 4, 451 day 7) derived from 1 experiment. Box plot boxes show the 25th and 75th percentile with the median, and whiskers indicate 1.5x the interquartile range. P values determined by two-sided unpaired t test (respectively p = 1.2e-7, p < 2.22e-16, p < 2.22e-16). f Representative immunofluorescence microscopy image for e), scale bar 10 µm.
