Lack of methylation changes in ATRA-treated APL patient blasts and PML-RARα–expressing murine hematopoietic progenitor cells. (A) Smoothed scatter plot of methylation values for ATRA-treated versus untreated APL patient blasts. Colors encode the density of points ranging from red (high density) to blue (low density). Cells were treated with 1μM ATRA or incubated with no treatment for 48 hours and analyzed by CD11b-FACS and cytospins for differentiation. A histogram plot of CD11b levels for mock-treated (blue) versus ATRA-treated blast cells (red) is also shown. (B) Analysis of methylation patterns in preleukemic PML-RARα knock-in mice. Gr1⁺ BM cells were extracted from heterozygous PML-RARα knock-in mice (n = 3) and age- and sex-matched C57BL6 wild-type mice (n = 3). Gr1⁺ sorting yielded a population consisting of granulocytes and hematopoietic progenitor cells. Final magnification was 100× (Axio Imager M1; Zeiss). Genomic DNA was extracted and subjected to RRBS analysis. The smoothed scatter plot compares the averaged methylation levels for knock-in and wild-type mice. No DMRs could be found between groups. (C) Retroviral transduction of hematopoietic progenitor cells with PML-RARα. BM was extracted from 12-week-old C57BL6 wild-type mice (n = 20) and sorted for Lin⁻ BM cells. Lin⁻ BM cells were then transduced with either PML-RARα or empty vector, sorted for green fluorescent protein expression, and then further cultivated for 10 days (cells from n = 10 per group). Genomic DNA was extracted on days 0, 5, and 10 and subjected to RRBS analysis. Cells were also analyzed using Wright-Giemsa–stained cytospins. Final magnification was 100× (Axio Imager M1; Zeiss). Smoothed scatter plots comparing methylation levels of PML-RARα and empty vector–transduced cells are shown. Indicated are the numbers of DMRs obtained by comparison of the respective samples.