Fig 2: Viability of TOV21G cells following HNF-1β, Claspin or USP28 siRNA transfection(A) After the transfection with Claspin, HNF-1β and control siRNAs was repeated twice (at day 1 and day 3), bleomycin (42 μM) was added to the TOV21G cells at day 4 and further incubated for 48 hours. (B) In a separate experiment, TOV21G cells were transfected with USP28, HNF-1β and control siRNAs at day 1 and treated with bleomycin at day 3. After 48 hours incubation, the cytotoxicity was determined by the trypan blue exclusion test. Cell viability (%) = living cells / total cells × 100%. The viability of TOV21G cells was evaluated using a trypan blue assay. Each condition was done in duplicates.

Fig 3: Knock down efficiency of HNF-1β (A), Claspin (B), and USP28 (C) siRNA. Western blot analysis assays revealed that the protein expression levels of HNF-1β (A), Claspin (B), and USP28 (C) were significantly decreased in the TOV21G si-HNF-1β, si-Claspin and si-USP28 groups when compared with the TOV21G si-Ctl group, with β-actin protein as the internal control. Knock-down was efficient in every experiment, resulting in a 70-85% reduction of the corresponding protein.

Fig 4: Effect of HNF-1β on the USP28 and USP29 protein expression after bleomycinWestern blot shows the protein levels of USP28 and USP29 in TOV21G cells transfected with si-HNF-1β or si-Ctl. Knockdown of endogenous HNF-1β led to a decrease in USP28, but not USP29, expression at the protein levels. Figure shows representative images from at least three independent Western blot assays.

Fig 5: Chemical trends in primary screening.a Heatmap representing potency and selectivity data for all 178 library compounds against 56 DUBs detected consistently over 80% of runs. b Electrophiles displayed diverse DUB hit rates. Acrylamides exhibited poor activity towards DUB catalytic cysteines. Insets adjacent to each bar indicate the number of hits and the total number of compounds containing each electrophile. c Presence and type of linker influenced compound activity and selectivity. d Individual warheads showed partially overlapping DUB target profiles. Venn diagram is colored by warhead, legend in figure. Bolded DUBs are hitherto untargeted. e, f Selected representative examples for multiple chemically similar hits for the same DUB from the screen. In the case of UCHL1, stringent requirements surrounding hydrogen bond acceptor position and amide orientation on the linker are highlighted in yellow. For VCPIP1, compounds were structurally similar except for the methylene group which moved along the backbone of the molecule (in blue). Moieties common across hits are highlighted for USP28 (in purple), USP3 (in red), and USP48 (in gray).