fig5

Figure 5. Identification of new USP14i. Medium-throughput screening for USP14i was performed using Ub-Rho110-G hydrolysis assay. (A) Primary screening results of 1,056 compounds at 20 µM; (B) Chemical structure of ARN12502; (C) Representative inhibition curve of ARN12502. The dotted red line indicates basal Ub-VS 26S activity (~20% of total measured activity). The dose-response curve is representative of three independent experiments; (D) USP14 activity in IGROV-1/Pt1 cells after treatment with USP14i. Deubiquitinases were labeled with HA-Ub-VS, followed by western blotting. Cells were treated with 100 µM IU1-47 or ARN12502 for 24 h. +, samples incubated with HA-Ub-VS; -, samples incubated without HA-Ub-VS. Vinculin served as loading control. The histogram reports percentage inhibition of USP14 activity (mean ± SD, three technical replicates); (E) Proteasome perturbation analysis in U2OS/pZS cells using flow cytometry after treatment with USP14i for 24 h. IU1-47 was used as reference. Values represent mean ± SD of three technical replicates. USP14: Ubiquitin-specific protease 14; USP14i: USP14 inhibitors; SD: standard deviation; HA-Ub-VS: hemagglutinin-ubiquitin-vinyl sulfone.