C527

In Vitro Deubiquitination Assays

Purified USP5 enzyme was purchased from Boston Biochem. UCH-L1 and UCH-L3 were as reported previously. USP12/46 was prepared in our laboratory as described. The in vitro enzymatic assays were performed as described previously using ubiquitin-AMC (Ub-7-amido-4methylcoumarin; Boston Biochem) 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 was measured by FluoStar Galaxy Fluorometer (BMG Labtech). For the Ub-vinylsulfone (VS) assay, the proteins were incubated with Ub-VS (Boston Biochem) at 0.5 μM final concentration for 45 min at 30 °C, followed by the immunoblotting analysis.

Cell lines

human U20S osteosarcoma cells, Hela cells

Preparation method

The solubility of this compound in DMSO is <2.93mg/mL. General tips for obtaining a higher concentration: Please warm the tube at 37℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.

Reacting condition

0.5, 1 and 2 μM; 24 h

Applications

In human U20S osteosarcoma cells, C527 promoted ID1 degradation in a dose-dependent way. In Hela cells, C527 increased the levels of Ub-FANCD2 and Ub-FANCI. Pre-treatment with C527 also enhanced the cytoxicity of DNA damaging agents including Mitomyin C and Camptothecin. C527 inhibited Camptothecin induced the Rad51 foci formation and reduced homologous recombination (HR) activity.

References:

1.Mistry H, Hsieh G, Buhrlage SJ et al. Small-molecule inhibitors of USP1 target ID1 degradation in leukemic cells. Mol Cancer Ther. 2013 Dec;12(12):2651-62. doi: 10.1158/1535-7163.MCT-13-0103-T. Epub 2013 Oct 15.