Bortezomib inhibits TPO-induced NF-κB activation both in vitro and in vivo. (A-B) Bortezomib inhibits TPO-induced NF-κB activation in vitro in (A) UT7/c-mpl cells and in (B) murine megakaryocytes. Cells were cytokine deprived for 16 hours prior to a 30 minute and 60 minute stimulation by either TNF-α (5 ng/mL) or GM-CSF (20 ng/mL), as positive controls, and by TPO (100 ng/mL) with or without bortezomib (2.5 ng/mL) pretreatment. Cells were stained with an Ab specific for the NF-κB subunit p65 (red) and with the chromatin dye DAPI (blue) in insets as described in Immunofluorescence and Western blot analysis, “Materials and methods.” Representative immunofluorescence pictures are shown. Original magnification, × 63. (C-D) Bortezomib inhibits TPO-induced NF-κB activation in vivo in TPO^high mice. (C) Nuclear levels of NF-κB subunits p65 and p50 in spleen cells from control mice, untreated TPO^high mice (vehicle), and treated TPO^high mice (bortezomib 0.5 mg/kg) were analyzed by Western blotting. The presence of Histone H1 was detected using an appropriate antibody and served as a loading control. (D) The amounts of the p65 and p50 NF-κB subunits were quantified using Pcbas Software and normalized with the level of Histone H1 per lane. (E-F) Plasma IL-1α and TPO levels were quantified using ELISA assays. (E) TPO^high mice overexpress similar plasma levels of TPO, and (F) bortezomib is able to decrease plasma concentration of IL-1α in a dose-dependent manner (P_(DE) ≤ .001). Results are presented as the mean plus or minus the SEM of 12 animals per experimental group (except for the “1 mg/kg” group that included only 6 surviving mice at week 8). Results of statistical analysis with the Wilcoxon test are as follows: treated (bortezomib 0.25 mg/kg, 0.5 mg/kg, and 1 mg/kg) versus untreated (Vehicle) mice; *P < .05. Bzb indicates bortezomib.