SAHA did not affect translation from cyclin D1 IRES site in MCL cells. Structure of the bicistronic reporter gene is shown (top panel). Plasmid contains both the R reniformis (Renilla) and firefly luciferase genes. Gene expression was driven by the simian virus 40 promoter (prom). Without the IRES sequence between the R reniformis and firefly luciferase, only R reniformis luciferase protein was translated. When an IRES sequence was inserted in front of the firefly luciferase gene, the firefly gene was also translated dependently on the IRES sequence. The reporter construct that contained cyclin D1 IRES region in front of the firefly luciferase gene was transfected into SP49 MCL cells. Transfected cells were treated either with or without SAHA (5 μM, 9 hours); cells were lysed and luciferase activity was measured. Firefly luciferease activity was normalized by R reniformis luciferase activity. Constructs without the IRES sequence were used as a negative control. Results represent mean value and SD of 3 experiments done independently. Results are normalized to the mean value of experiments using the IRES (+) construct and SAHA.