Preparations of 26S proteasomes (1 nM) are pretreated with DMSO, VLX1570, or b-AP15 for 2 min in assay buffer (25 mM Tris, 5 mM MgCl₂, 10% glycerol, 0.05 mg/mL BSA, 2 mM ATP, and 1 mM DTT) before addition of Ubrhodamine. Fluorescence is monitored at 37°C using Ex/Em = 490 nm/520 nm to read data every 10 second for 30 min using a TECAN infinite 200 instrument. For UbVS labeling of KMS 11 cells, cell pellets are lysed from control or treated cells with buffer (50 mM HEPES pH 7.4, 250 mM sucrose, 10 mM MgCl₂, 2 mM ATP, 1 mM DTT) on ice for 30 min and removed debris by centrifugation. Twenty five μg of protein is labeled with 1 μM UbVS for 30 min at 37°C. Samples are resolved by SDS-PAGE and subjected to immunoblotting. For UbVS labeling of proteasomes, purified 19S proteasomes (50 nM) are pretreated with DMSO, VLX1570, or b-AP15 (50 μM) for 10 min at room temperature, followed by labeling with 1 μM HA-UbVS for 30 min at 37°C and by immunoblotting^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Cell viability is monitored by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. For the MTT assay, cells are suspended at 5 × 10⁵ cells/mL, and 100?μL aliquots are dispended into 96-well microtiter plates and exposed to drugs using DMSO as control. At the end of incubations, 10?μL of a stock solution of 5?mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), is added into each well, and the plates are incubated 4?hours at 37°C. Formazan crystals are dissolved with 100?μL of 10% SDS/10?mM HCl solution overnight at 37°C. Since MTT assays are affected by mitochondrial activity, and since OXPHOS is affected by VLX1570, the acid phosphatase method49 are used to determine cell viability in some experiments. After washing twice with PBS, cells are lysed in 100?μL of 0.1?M sodium acetate, 0,1% Triton X-100, p-nitrophenylphosphate and incubated for 90?min at 37°C. At the end of the incubation, 10?μL NaOH is added to each well and A405 is determined^[2].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Animal experiment is performed with a sample size of 7 per group, 80% power at the 5% significance level to detect a difference in means of 1800 mm³ between the 2 groups. For percentage change in IgM from baseline, with a sample size of 7 per group, 80% power at the 5% significance level is calculated to detect a difference in means of 450% between the 2 groups. Fourteen female NOD/SCID mice (6-8 weeks of age) are subcutaneously implanted with 1× 10⁶ luciferase labeled RPCI-WM1 cells (Luc-RPCI-WM1), which are allowed to grow till a bioluminescent signal is observed by IVIS imaging (Day 20). On day 21, mice are randomized into 2 groups (n=7 each), with one group receiving vehicle (cremaphor+PEG+Tween) and the other receiving VLX1570 at 4.4 mg/kg via intraperitoneal injection. The investigator is not blinded to the group allocation. Both groups are respectively treated with either vehicle or VLX1570 every alternate day for 22 days. Sizes of the tumors is measured every 3- 4 days using direct caliper measurements, and volume of the tumorsis calculated using the formula (width)² × length/2. Bioluminescent tumor imaging is performed with the Xenogen imaging system on Days 0, 20, 30, 36 and 43 post-tumor implantation. Blood from mice is collected on the same days by submandibular venous puncture, with sera subsequently separated for quantification of human IgM levels’ using ELISA. On Day 44, mice are sacrificed, and final tumor volume is measured in control and treatment arms. All images are obtained using a Canon D40 digital camera. No specific criteria for inclusion/exclusion are used as all mice formed tumors and are therefore included into the study^[3].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Wang X, et al. Synthesis and evaluation of derivatives of the proteasome deubiquitinase inhibitor b-AP15. Chem Biol Drug Des. 2015 Nov;86(5):1036-48.  [Content Brief]

  [2]. Wang X, et al. The proteasome deubiquitinase inhibitor VLX1570 shows selectivity for ubiquitin-specific protease-14 and induces apoptosis of multiple myeloma cells. Sci Rep. 2016 Jun 6;6:26979.  [Content Brief]

  [3]. Paulus A, et al. Coinhibition of the deubiquitinating enzymes, USP14 and UCHL5, with VLX1570 is lethal to ibrutinib- or bortezomib-resistant Waldenstrom macroglobulinemia tumor cells. Blood Cancer J. 2016 Nov 4;6(11):e492.  [Content Brief]