Time course of Noxa activation at protein and mRNA levels. Cells from Granta-519 (A), Jeko (B), and primary cells from MCL patient 1 (C) were treated with 20 nM bortezomib, and primary cells from MCL patient 2 (D), with 50 nM bortezomib. RNA and protein were isolated after 1, 2, 4, 6, and 14 hours of incubation. Noxa mRNA levels were evaluated at these time points by quantitative RT-PCR (Taqman technology) using GUS as housekeeping gene. mRNA-expression levels are given in arbitrary units, using cDNA from untreated cells as reference control. Noxa protein levels were evaluated by Western blotting using α-tubulin as equal loading control. Relative protein quantification was analyzed with Image Gauge software from Fujifilm LAS 3000 chemiluminescence detector. The percentage of loss of ΔΨ_m was determined by DiOC₆(3) staining, followed by FACs analysis.