Figure 2
Figure 2. CNS cocultures cause dormancy in Mer-expressing leukemic cell lines and protect from MTX toxicity and shRNA-mediated Mer knockdown in 697 cells reverses this phenotype. (A) 697 control cells and 697 cells taken out of a 48-hour U343 coculture were seeded at equal densities under standard culture conditions. Viable cells were counted at the time points indicated. (B) MTX treatment in coculture with U343 cells. 697, RS4;11, and HL-60 cells were treated with 100 nM MTX or DMSO (veh) for 48 hours alone (−) or in cocultures with U343 cells (+). MHH-CALL3 cells were treated for 96 hours. The percentage of dead leukemic cells was determined by trypan blue exclusion. (C) Viable cells in NRA cocultures. Leukemic cells were cultured alone (−) or in coculture with NRAs (+) for 48 hours. The number of viable leukemic cells was determined by trypan blue exclusion. (D) BrdU incorporation and 7-AAD staining in 697 cells cultured alone or in 48-hour cocultures with NRAs. Phases of the cell cycle are depicted as indicated. (E) MTX treatment in NRA coculture. 697, (Merhigh) and HL-60 (Merlow) cells were treated with 100 nM MTX or DMSO (veh) for 48 hours alone (−) or in cocultures with NRAs (+). The percentage of dead leukemic cells was determined by trypan blue exclusion. (F-G) Efficiency of the Mer knockdown by shRNA, as determined by qPCR (F) and FACS staining (G). (H) 697shGFP and 697shMer cells were cultured alone (−) or in coculture with U343 (+) for 72 hours in serum-free media. The number of viable leukemic cells was determined by trypan blue exclusion. (I) Growth curves after coculture. 697shGFP and 697shMer cells taken out of a 48-hour U343 coculture were seeded at equal densities in standard culture conditions. Viable cells were counted at the time points indicated. (J) MTX in U343 coculture. 697shGFP and 697shMer cells were treated with 100 nM MTX or DMSO (veh) for 48 hours alone (−) or in cocultures with U343 cells (+). The percentage of dead leukemic cells was determined by trypan blue exclusion. *P < .05; **P < .01; ***P < .001.

CNS cocultures cause dormancy in Mer-expressing leukemic cell lines and protect from MTX toxicity and shRNA-mediated Mer knockdown in 697 cells reverses this phenotype. (A) 697 control cells and 697 cells taken out of a 48-hour U343 coculture were seeded at equal densities under standard culture conditions. Viable cells were counted at the time points indicated. (B) MTX treatment in coculture with U343 cells. 697, RS4;11, and HL-60 cells were treated with 100 nM MTX or DMSO (veh) for 48 hours alone (−) or in cocultures with U343 cells (+). MHH-CALL3 cells were treated for 96 hours. The percentage of dead leukemic cells was determined by trypan blue exclusion. (C) Viable cells in NRA cocultures. Leukemic cells were cultured alone (−) or in coculture with NRAs (+) for 48 hours. The number of viable leukemic cells was determined by trypan blue exclusion. (D) BrdU incorporation and 7-AAD staining in 697 cells cultured alone or in 48-hour cocultures with NRAs. Phases of the cell cycle are depicted as indicated. (E) MTX treatment in NRA coculture. 697, (Merhigh) and HL-60 (Merlow) cells were treated with 100 nM MTX or DMSO (veh) for 48 hours alone (−) or in cocultures with NRAs (+). The percentage of dead leukemic cells was determined by trypan blue exclusion. (F-G) Efficiency of the Mer knockdown by shRNA, as determined by qPCR (F) and FACS staining (G). (H) 697shGFP and 697shMer cells were cultured alone (−) or in coculture with U343 (+) for 72 hours in serum-free media. The number of viable leukemic cells was determined by trypan blue exclusion. (I) Growth curves after coculture. 697shGFP and 697shMer cells taken out of a 48-hour U343 coculture were seeded at equal densities in standard culture conditions. Viable cells were counted at the time points indicated. (J) MTX in U343 coculture. 697shGFP and 697shMer cells were treated with 100 nM MTX or DMSO (veh) for 48 hours alone (−) or in cocultures with U343 cells (+). The percentage of dead leukemic cells was determined by trypan blue exclusion. *P < .05; **P < .01; ***P < .001.

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