Chemical and molecular genetic validation of the adenosine receptor subtype A2A, PDE3B and PDE7A as a novel drug targets for multiple myeloma. (A) MM.1S cells were incubated with 3.4μM trequinsin, 0.77μM Chloro-IB-MECA, or both drugs in the absence or presence of 89nM DPCPX (A1 antagonist), 78nM SCH 58261 (A2A antagonist), 89nM MRS 1754 (A2B antagonist), and 87nM MRS 1523 (A3 antagonist). Cells were seeded in wells of a 384-well plate, incubated with drugs for 72 hours, and effects on proliferation determined using ATP lite. (B) MM.1R cells were electroporated with 50nM control siRNA (siCON) or siRNA targeting the A1, A2A, A2B, or A3 adenosine receptor. Forty-eight hours later, cells were exposed to 510nM HE-NECA or 320nM Chloro-IB-MECA and incubated an additional 72 hours before the addition of ATP lite. At the time of drug addition, as determined by reverse transcription–PCR, target RNA knockdown was 88% for the adenosine A1 receptor, 76% for A2A, 37% for A2B, and 62% for the A3 adenosine receptor subtype. The results are from experiments performed in duplicate. MM.1R cells were electroporated with control siRNA (siCON) or siRNA targeting (C) PDE3B or (D) PDE7A. Cells not receiving PDE siRNA were electroporated with the control siRNA siCON. Forty-eight hours postelectroporation, cells were seeded in 384-well plates, treated with 250nM roflumilast (PDE4i), 2.3nM HE-NECA (A2A agonist), or the combination of both drugs and incubated an additional 72 hours followed by the addition of ATP lite. As determined by RT-PCR, at the time of drug addition, target RNA knockdown was 64% for PDE3B and 60% for PDE7A. All results are from experiments performed in duplicate.