Combined treatment of ANBL-6 cells with PD98059 and Sant7 leads to an apoptotic response in the absence and in the presence of BMSCs. (A) Western blots showing the phosphorylation status of ERK1, ERK2, and STAT3 (Tyr705) in ANBL-6 cells cultured for 5 days with or without BMSCs, and with combinations of 50 μM PD98059 and 50 μg/mL Sant7. Staining for ERK1,2 or STAT3 served as loading controls. Low basal levels of ERK phosphorylation were strongly augmented in coculture with BMSCs, and PD98059 effectively inhibited this activation (lanes 6 and 7). Treatment with Sant7 alone entailed a considerable down-regulation as well (lane 8). Sant7 efficiently inhibited IL-6– or BMSC-induced phosphorylation of STAT3 (lane 8). (B) Survival of ANBL-6 cells kept in medium with IL-6 or in coculture with BMSCs, and exposed to combinations of PD98059 (50 μM) and Sant7 (50 μg/mL). PD98059 alone was ineffective, and the 50% reduction in viability achieved with Sant7 was largely lost when the cells were cocultured with BMSCs. The combination of both drugs led to near-complete cell death in both settings. (C) Survival of ANBL-6 cells in medium with IL-6 or in coculture with BMSCs, with or without addition of 50 μM PD98059 and with increasing concentrations of Sant7. Apoptosis was assayed after 5 days. Sant7 was moderately effective at decreasing the viability of ANBL-6 cells, and addition of PD98059 strongly enhanced this effect. Applied alone, PD98059 reduced the viability by some 20%. Data shown are the means of 3 independent experiments. Statistical analysis (B) revealed significant (^*) or very significant differences (^**) as indicated. Error bars denote the range of values derived from at least 3 independent experiments.