NPI-0052 inhibits Akt pathway and synergizes with bortezomib in inhibiting Akt and 20S proteasome activities. (A) BCWM.1 cells were cultured with NPI-0052 (2.5-20 nM) for 6 hours. Whole-cell lysates were subjected to Western blotting using anti–p-Akt, -Akt, –p-GSK3α/β, –p-S6R, –p-ERK, and –α-tubulin antibodies. (B) In vitro Akt kinase assay. BCWM.1 cells were cultured with control media or NPI-0052 (2.5-20 nM) for 6 hours. Whole-cell lysates were immunoprecipitated with anti-Akt antibody. Then the immunoprecipitate was washed and subjected to in vitro kinase assay according to the manufacturer's protocol. Western blotting used anti–p-GSK3α/β and anti-Akt antibodies. (C) BCWM.1 cells were transduced with Akt shRNA for 48 hours. Mock indicates control plasmid. BCWM.1 transfected cells or BCWM.1 control cells were treated with NPI-0052 (2.5-20 nM) for 48 hours. Cytotoxicity was assessed by MTT assay. Whole-cell lysates were subjected to Western blotting using anti–p-Akt, -Akt, and –α-tubulin antibodies (insert in C). (D) BCWM.1 cells or primary CD19⁺ tumor cells from 2 patients with WM (Ei-iii) were incubated for 4 hours in the presence of diluent or 10 nM NPI-0052, bortezomib 10 nM, or bortezomib + NPI-0052. The chymotrypsin-like (CT-L), trypsinlike (T-L), and caspaselike (C-L) activity of the 20S proteasome of BCWM.1 was determined by measurement of fluorescence generated from the cleavage of the fluorogenic substrates suc-LLVY-amc, boc-LRR-amc, and z-LLE-amc, respectively.