Inhibition of Mer expression increases cell death and promotes apoptotic signaling pathway activation in response to chemotherapy. Apoptotic and dead cells were identified by flow cytometric analysis of cells stained with YO-PRO-1 iodide and PI. (A) Representative 697 flow cytometry profiles after treatment with methotrexate (MTX) are shown. The percentages of cells in early apoptosis (YO-PRO-1 positive, right lower quadrant), cells in later stages of apoptosis and necrosis (PI positive, upper quadrants), and live cells (left lower quadrant) are shown. Cultures of 697 and REH shControl, shMer1, and shMer4 cells were exposed to (B) 30 nM methotrexate, (C) 0.2 nM vincristine (VINC), (D) 50 nM (697) or 10 µm (REH) dexamethasone (DEX), or (E) 3 IU/mL l-asparaginase (L-ASP) or equivalent vehicle (phosphate-buffered saline [PBS] for methotrexate and vincristine; ethanol for dexamethasone and l-asparaginase) for 48 hours. Drug treated denoted as “+”, and vehicle treated denoted as “−”. Mean values and SEs derived from at least 3 independent experiments are shown. In response to methotrexate and vincristine, both REH and 697 Mer knockdown cells (shMer1 and shMer4) exhibited significant synergistic increases in the total percentage of apoptotic and dead cells compared to drug-treated shControl cells. In response to dexamethasone and l-asparaginase treatment, 697 cells exhibit significant induction of apoptosis. REH shMer cells exhibited a trend toward increased sensitivity in response to l-asparaginase, although there was no change in response to dexamethasone. *P < .05; **P < .01. (F) Whole-cell lysates were prepared from 697 and REH wild-type, shControl, shMer1, and shMer4 cells treated with methotrexate for 48 hours, and PARP, caspase 3, and caspase 8 were detected by immunoblot analysis. Representative 697 immunoblots are shown.