MEK signaling regulates MAF transcription. (A) Myeloma cell line LP1 was treated with small-molecule kinase inhibitors targeting major cell signaling cascades at the indicated concentrations. MAF mRNA was measured by quantitative RT-PCR. (B) LP1 MM cells were transduced with a TET-inducible MMSET shRNA construct, selected, and MMSET shRNA was induced for the indicated times. Protein lysates were analyzed by Western blot. ERK phosphorylation and FOS expression were decreased in these cells. (C) MAF-expressing myeloma cell lines were treated with MEK inhibitor U0126 (10μM), and MAF mRNA was measured by real-time quantitative PCR. (D) MAF and target gene mRNA were measured by quantitative RT-PCR after treatment with a second MEK inhibitor, AZD6244 100nM, for the indicated times. (E) MM cells transduced with 2 individual TET-inducible MEK1 shRNA construct, selected, and MEK1 shRNA was induced for the indicated times. Protein lysates were analyzed by Western blot to confirm MEK1 protein decrease after shRNA-mediated knockdown. (F) MAF expression was quantified by quantitative RT-PCR after MEK1 depletion with each shRNA construct in 2 MM cell lines. (G) MAF and target gene mRNA expression were measured by quantitative RT-PCR after MEK1 depletion. Error bars represent mean plus or minus SD.