Epigenetic Library Screening.

A focused library of 96 epigenetic modifiers (Cayman Chemicals) was screened for the ability to induce differentiation of JK-1 cells. Undifferentiated JK-1 cells were obtained by FACS by gating for an α-GypA–FITC–negative population. In the first experimental run, cells were diluted to 4.0 × 10⁴ cells per well in 200 μL JK-1 growth medium in 96-well flat-bottomed plates (Falcon), and epigenetic modifiers were added to 10 μM or 1 μM final concentration using the robotics facility at the Institute of Chemistry and Cell Biology at Harvard Medical School. Cells were grown for 5 d under standard growth conditions before harvesting. In the second experimental run, cells were diluted to 8.0 × 10³ cells per well with the same two concentrations (10 μM and 1 μM) of epigenetic modifiers. Half of the cells were harvested at day 7 post setup, and the medium was refreshed for the remainder of the cells, which were allowed to grow until day 14 post setup. The harvested cells were stained with α-GypA–FITC, and the level of GypA was measured by flow cytometry on a Miltenyi MACSQuant flow cytometer (Miltenyi). The GypA-high:GypA⁻ ratio was calculated from plots of SSC vs. α-GypA–FITC (Fig. S2 A and B) for each compound, and the values were normalized to the highest ratio for each concentration of each experimental run. The data were clustered using Gene Cluster version 3.0 (82) by hierarchical clustering with a Euclidean distance similarity metric and complete linkage. The data were visualized using TreeView version 1.1.6r4 (83).