Signaling of A3AR is responsible for GC-induced ERK1/2 phosphorylation and enhanced resistance to apoptosis. Monocytes were stimulated for 4 and 16 hours with 100nM DEX (■) or left untreated (□). Quantitative RT-PCR was performed to confirm the up-regulated expression of A3AR (A). Plots shows relative N-fold regulation ± SEM compared with control monocytes (n = 3). Monocytes were pretreated in the presence of 0.5μM A3AR antagonist MRS1220 for 30 minutes before stimulation with DEX (GC) or medium as control (Co). After 16 hours, cells were stimulated with 200nM STS (■) or left untreated (□). After 6 hours, apoptosis was measured by annexin V staining (B). In parallel, protein levels of phosphorylated and total ERK were determined by Western blot analysis (C). Monocytes were preincubated in the presence of 100μM Boc-FLFLF inhibitor for 30 minutes before 16 hours stimulation with DEX and subsequently challenged to apoptosis induced by STS (■) or left untreated (□). After 6 hours, amounts of apoptotic cells were measured by annexin V staining (D). Data show mean ± SEM (n = 3). Simultaneously, cells were lysed, and immunoblotting was performed using anti-phospho ERK and total ERK antibodies (E). Monocytes were treated for 4 and 16 hours with 100nM DEX. Changes in expression of c-myc were confirmed using qRT-PCR (F). After 16 hours of stimulation with 100nM DEX, ChIP was performed to check c-Myc binding to the promoter of SAP30 and Myc genes, and promoter binding was quantified by PCR (G).