Fig. 6: Identification and characterization of HYP as a USP18 inhibitor that directly targets the USP18 IBB1 domain. | Cell Death & Disease

Fig. 6: Identification and characterization of HYP as a USP18 inhibitor that directly targets the USP18 IBB1 domain.

From: Targeting USP18 overcomes acquired resistance in hepatocellular carcinoma by regulating NCOA4 deISGylation and ferroptosis

Fig. 6

A The flow diagram for USP18 inhibitor screening. B Computational model and interactions of the USP18 IBB1 domain and HYP. C, D Biolayer interferometry (BLI) determination of the binding kinetics between HYP and USP18 WT (C) or USP18 IBB1 MUT (D). BLI response profile for HYP at different concentrations with sensor-immobilized USP18 WT or USP18 IBB1 MUT. KD, equilibrium dissociation constant; Kon, association rate constant; Koff, dissociation rate constant. E SDS-PAGE indicating the purification of the USP5, USP14, USP16, and USP18 proteins. F ISG15-AMC hydrolysis experiment. The enzymatic activity of USP5, USP14, USP16, or USP18 in the presence of increasing concentrations of HYP. G Protein expression of NCOA4 in HCC-SR cells treated with indicated concentrations of HYP for 24 h. The band intensities were quantified using Image J and normalized to the control cells treated with DMSO. H Expression of NCOA4 ubiquitination and ISGylation in anti-NCOA4 immunoprecipitation and whole-cell lysates (input) derived from HepG2-SR cells treated with indicated concentrations of HYP for 24 h and MG132 (10 μM) for 4 h. *, heavy chain. I, J Colony formation assay (I) and CCK-8 (J). The impact of HYP on the susceptibility of HepG2-SR cells toward sorafenib treatment (means ± SEM, **p < 0.01, one-way ANOVA test). K HepG2-SR cells were transfected with siNC or siUSP18 for 48 h and treated with sorafenib and/or 40 μM HYP for 24 h. The cell viability was measured by CCK-8 assay (means ± SEM, *p < 0.05, one-way ANOVA test).

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