Cellular UbG76V-GFP accumulation assay

Ub^G76V-GFP/ODD-Luc HeLa cells⁹⁸ were plated as 4500 cells per well per 30 μL in 384 well plate (Greiner 781091). Clear DMEM containing 2.5% FBS, 1% L-glutamine, and 1% penicillin-streptomycin was used as an assay medium. The next day, cells were treated with the compounds (serial 2-fold dilutions with a starting concentration of 42, 10.5, or 4.2 μM, eight concentrations) and incubated for 6 h. ImageXpress high-content confocal microscope (Molecular Devices, LLC., San Jose, CA) was used to image cells at GFP channel. The percentage of Ub^G76V-GFP accumulation (%Acc) is calculated using the following formula:

%Acc = (GFP signal of test compound – GFP signal of DMSO)/(GFP signal of 5 μM NMS-874 – GFP signal of DMSO) * 100.

The IC₅₀ values were calculated by fitting the %Acc from four replicates with various concentrations of compounds using GraphPad Prism 7.0 (GraphPad, Boston, MA).