Inhibition of Hsp70 down-regulates Tcl1 protein in CLL. (A) CLL patient samples were treated for 12 hours with mock or Myr (50μΜ) or 17AAG (5μΜ) and then analyzed for Tcl1 and Gapdh by Western blotting. The numbers above the blots indicate the intensity of the band expressed as ratio gene product TCL1/GAPDH and normalized to DMSO. (B) Myr increases TP53 promoter activation, blocking Hsp70 activity. HEK-293A cells were cotransfected for 24 hours with 50 ng of TP53-Luc reporter and 50 ng of pRL-TK Renilla reporter constructs. In addition, 0.75 μg of CMV5-empty vector, or 0.75 μg of CMV5-TCL1 WT were used. Five nanograms of pFC-MEKK was added. Cells were treated with 10μM Myr for 12 hours, where indicated. Data are representative of 3 independent experiments. Data are mean ± SEM of 3 independent experiments, and each is measured in triplicate (*P < .05). (C) Tcl1 activates NF-κB–dependent transcription. HEK-293A cells were cotransfected for 24 hours with 50 ng of pNF-κB-Luc reporter and 50 ng of pRL-TK Renilla reporter constructs. In addition, 0.75 μg of CMV5-empty vector, or 0.75 μg of CMV5-TCL1 WT were used. Five nanograms of pFC-MEKK was added. Cells were treated with 10μM Myr for 12 hours, where indicated. Data are representative of 3 independent experiments. Data are mean ± SEM of 3 independent experiments, and each is measured in triplicate (*P < .05).