The in vitro enzymatic assays are performed using ubiquitin-AMC (Ub-7-amido-4methylcoumarin) as a substrate in a reaction buffer containing 20 mM HEPES-KOH (pH 7.8), 20 mM NaCl, 0.1 mg/mL ovalbumin, 0.5 mM EDTA and 10 mM dithiothreitol. The fluorescence is measured by FluoStar Galaxy Fluorometer. For the Ub-vinylsulfone (VS) assay, the proteins are incubated with Ub-VS at 0.5 μM final concentration for 45 min at 30°C, followed by the immunoblotting analysis^[1].

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

Leukemic cell lines are grown in RPMI 1640 medium supplemented with 10% fetal bovine serum and penicillin/streptomycin. Hela cells and U2OS cells are grown in DMEM supplemented with 10% fetal bovine serum and penicillin/streptomycin. USP1 inhibitor C527 and its derivatives (e.g., SJB2-043) are synthesized and the purity is validated by high-performance liquid chromatography. Primary human AML patient samples are collected from DFCI leukemia program under the approval of appropriate protocols. Cells are treated with DMSO or USP1 inhibitors (e.g., SJB2-043) in appropriate medium for 24-72 hrs. The viable cell counts are determined using Trypan blue staining, Cell TiterGlo reagent or MTT assay. The apoptotic cells are detected using AnnexinV and 7AAD staining using flow cytometry. For Benzidine staining, the cells are washed twice with PBS and resuspended in 45 μL of PBS + 5 μL of Benzidine stain solution (0.2% in 0.5 M glacial acetic acid, 3% H₂O₂). After 45 min incubation at room temperature, the Benzidine positive cells are detected by light microscopy^[1].

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

• [1]. Mistry H, et al. Small molecule inhibitors of USP1 target ID1 degradation in leukemic cells. Mol Cancer Ther. 2013 Dec;12(12):2651-62.  [Content Brief]