Figure 4. VLX1570 induces proteasome-associated polyubiquitin accumulation and apoptosis of multiple myeloma cells.

(a) MM cells were exposed to 0.5 μM VLX1570, 0.5 μM b-AP15 or 50 nM bortezomib (BZ) for 6 or 18 h, followed by immunoblotting for active caspase-3, Ub-K48, HSP70B  ´(HSPA6), Hmox-1, phospho-JNK, JNK and β-actin. RPMI8826 and KMS11 data are from the same filter, OPM-2 from a separate (dashed lines were introduced for clarity). (b) Determination of cell death induction after exposure to 0.5 μM VLX1570 or vehicle for 0, 6 or 18 h. Cells were stained with FITC-conjugated annexin V and propidium iodide and processed by flow cytometry. The percentage of viable, early apoptotic and late apoptotic were quantified. Results shown are mean values of triplicate measurements. (c) Estimation of cell division based on dilution of carboxyfluorescein succinimidyl ester (CFSE) membrane staining during cell division. Cells were exposed to 0.25 μM or 0.5 μM VLX1570 or DMSO as a control for 72 h. The fraction of cells in different generations was determined by flow cytometry analysis. (d,e) Analysis of the effects of VLX1570 on the sedimentation profiles of polyubiquitinated proteins. Cell lysates from control or VLX1570-exposed OPM-2 (c) or KMS-11 cells (d) were subjected to glycerol gradient centrifugation. Gradient fractions were collected (1 = top; 13 = bottom) and subjected to immunoblotting using antibodies to Ub-K48, PSMD14 (19S subunit), or PSMB5 (20S subunit). Note the accumulation of polyubiquitin on 26S proteasomes after 6 hours of drug exposure.