Fig. 3: YL1004 inhibits PLpro deubiquitinating and deISGylating activities.
a IC50 curves of YL1004 in inhibiting PLpro hydrolysis of Ub-AMC (left) and ISG-AMC (right), n = 3. b In-cell deubiquitinating activities of PLpro, HEK293T cells were transfected with plasmids encoding PLpro-Flag, Ub-HA, and indicated compounds alone or in combination. Then cell lysates were subjected to immunoblotting with anti-HA, anti-Flag, and anti-GAPDH antibodies. c HEK293T cells were treated with or without IFN-α for 48 h. Then the cell extracts were incubated with PLpro and indicated compounds, followed by immunoblotting analysis with anti-ISG15 and anti-PLpro antibody. d Counter screening of YL1004 in inhibiting common human DUBS hydrolysis of Ub-AMC (n = 2). e In cell specificity of YL1004 for PLpro over human DUBs. Lines 1 ~ 6: An anti-HA Western blot of lysed HEK293T cells treated with HA-Ub-VS in the presence of N-ethyl-maleimide (NEM, positive control inhibitor) or test compounds. Lines 7 ~ 12: PLpro was added to cell lysate before covalent modification by HA Ub-VS, showing that YL1004 eliminated PLpro-based modification. f–i YL1004 antagonized PLpro suppression on NF-κB (f), ISRE (g), IFN-β (h) or IRF3 (i) activation. Dual-luciferase reporter gene assays were performed in HEK293T cells (n = 3). Data in (a), (d), and (f-i) were shown as mean ± sd. Experiments (b), (c), and (e) were repeated three times independently with similar results. The samples were derived from the same biological source, and the corresponding gels and blots were processed in parallel to ensure consistency.
