Validation of screening results by quantitative DNA methylation analysis (MassARRAY). (A) Heatmap showing the average methylation levels in 40 gene-specific amplicons (columns) and individual PU.1-kd and PU.1-wt animals (rows). Methylation levels range from 0% (light green) to 100% (dark blue). Grey boxes represent missing values. The bar to the left of the heatmap indicates different disease stages (black, preleukemic; blue, early leukemic; red, late leukemic; green, PU.1-wt). Unsupervised clustering discriminates PU.1-kd and PU.1-wt animals. (B,C) DNA methylation levels of Fzd5 (B) and Fzd8 (C). Average amplicon methylation is shown for different animals of the different stages. The black bar represents the median methylation within one stage. Mann-Whitney U test was used to test for differences between the different disease stages (*P < 0.05, **P < 0.01, ***P < 0.001). (D,E) Methylation levels (heatmaps on top, bar graphs below) of Fzd5 (D) and Fzd8 (E) amplicons in sorted cells of PU.1-wt animals and two groups of preleukemic PU.1-kd animals, KD1 and KD2. The sorted cells comprise LSKs (lineage-negative, Ska1-positive, c-kit-negative cells), CMPs (common myeloid progenitor cells), GMPs (granulocyte-macrophage progenitor cells) and MEPs (megakaryocyte-erythroid progenitor cells). The heatmaps display single CpG units (columns) of PU.1-kd or PU.1-wt animals (rows). Methylation values range from 0% (light green) to 100% (dark blue). The bar graphs show average methylation (y-axis) of the different amplicons. In (D), analysis of KD2-GMP failed, indicated by grey CpG units in the heatmap and missing value in the bar graph.