PRAME silencing in primary CML progenitor cells increases myeloid colony formation. (A) shPRAME-transduced cells (left) compared with shControl cells (shGFP, right) from 3 CML blast crisis patients exhibited increased colony formation on agarose both in the absence and presence of ATRA at 0.01 and 0.1 μM. Colonies arising from shPRAME and shControl cells exposed to 0.01 μM ATRA from 1 patient are shown. (B) To confirm colony morphology after exposure to GM-CSF and G-CSF individual colonies (i) were plucked and stained with Wright-Giemsa. Colony types were predominately granulocytic (CFU-G, ii) or monocytic (CFU-M, iii), but CFU-GMs (iv) were also seen. (C) shPRAME silenced CML cells compared with shControl cells from 3 patients demonstrated increased numbers of CFU-GMs, CFU-Gs, and CFU-Ms on days 0 and 2. The mean colony numbers from independent experiments performed in triplicate are shown. The numbers represented on the y-axis indicate the numbers of progenitor cells in culture on each experimental day that gave rise to a colony. (D) For 2 patients, sufficient cells were available to assess CFU formation after exposure to ATRA at 0.01 μM and 0.1 μM. Similar to the phenotype seen in the absence of ATRA, shPRAME cells compared with shControl cells formed increased numbers of CFU-GM, CFU-G, and CFU-M colonies. Day 2 is shown; day 4 shows the same increase in CFUs in shPRAME cells compared with shControl cells (included in calculations of statistical significance). Furthermore, the same differences were observed in 1 patient whose cells were treated with 0.001 μM ATRA. The mean numbers of colonies are shown from independent experiments performed in triplicate. The numbers represented on the y-axis indicate the numbers of cells in culture on a particular experimental day that gave rise to a colony.