Fig 2: Increased BRCA1 expression inhibits ISGylation and decreases ISG15, USP18, and EGFR protein levels. (A) Representative Western blot showing free and conjugated ISG15 levels in OE-Mock and OE-BRCA1 cells in the presence and absence of 24-hour treatment with 1000 U/ml IFNß. Levels of HSP90 are shown as a loading control. LE=long exposure; SE=short exposure. (B and C) Comparison of free ISG15 (B) and ISG15 conjugates (C) in OE-Mock and OE-BRCA1 cells in the presence or absence of IFNß treatment. Bars represent the mean ± SEM of six independent experiments relative to levels measured in vehicle-treated OE-Mock cells. *P<.05; **P<.01; ***P<.001, t test. (D) Representative Western blot showing USP18 levels in OE-Mock and OE-BRCA1 cells in the presence and absence of 24-hour treatment with 1000 U/ml IFNß. (E and F) Comparison of USP18 levels on OE-Mock and OE-BRCA1 cells in the absence (E) or presence (F) of IFNß treatment. Bars represent the mean ± SEM of three independent experiments relative to levels measured in OE-Mock cells. Levels of USP18 following IFNß treatment were determined on short exposure blots. *P<.001, one-sample t test. (G) Representative Western blot showing levels of EGFR in OE-Mock and OE-BRCA1 cells. (H) Comparison of EGFR levels normalized to HSP90. Bars represent the mean ± SEM of four independent experiments relative to levels measured in OE-Mock cells. *P<.02, one-sample t test.

Fig 3: Effects of USP18 and USP20 depletion on viral lytic replication in SCC9 cells(A) Examination of si-USP18 and si-USP20 knockdown efficiency. Error bar, mean ± SEM. (B) Virus yields in USP18 or USP20-depleted SCC9 cells. (C and E) Accumulation of viral proteins in USP18 or USP20-depleted SCC9 cells. ß-actin serves as a loading control. In addition, the band density of viral protein by gray analysis. Relative density was normalized to ß-actin. (D and F) mRNA accumulation levels of viral ICP8 and VP16 in SCC9 cells.

Fig 4: USP18 alone is not sufficient to induce desensitization Knockdown of USP18 results in sustained signaling. USP18 knockdown efficiency determined by quantitative immunoblotting of cytoplasmic lysates of Huh7.5 transfected with USP18 siRNA relative to parental Huh7.5 cells and Huh7.5 cells transfected with non-targeting siRNA. Cells were stimulated with 1,400 pM IFNa for 24 h. Error bars represent 1s confidence intervals estimated from biological replicates (N = 6 to N = 10) (left). Model fit and experimental data of Huh7.5 cells transfected with control siRNA or USP18 siRNA are shown. Cells were transfected with USP18 siRNA or control siRNA 1 day prior to growth factor-depletion. Next, cells were prestimulated with 1,400 pM IFNa (yellow background) and stimulated with 1,400 pM IFNa at 24 h or untreated (white background). IFNa-induced phosphorylation of STAT1 and STAT2 and induction of USP18 and tSTAT1, comprising both phosphorylated and unphosphorylated STAT1 protein, are displayed. Experimental data were obtained by quantitative immunoblotting using chemiluminescence and CCD camera-based device (ImageQuant). For model purposes, data in control siRNA and untransfected Huh7.5 are combined to one condition. Data from multiple time courses scaled together are displayed as filled circles with errors representing 1s confidence intervals estimated from biological replicates (N = 2 to N = 10) using a combined scaling and error model. Lines represent model trajectories.Overexpression of Huh7.5 is not sufficient to explain desensitization. Induced expression of USP18 after treatment of Huh7.5-TetON-USP18 and Huh7.5-TetON-control cells with doxycycline for 24 h in comparison with parental Huh7.5 cells treated with 1,400 pM IFNa for 24 h. Analysis was performed by quantitative immunoblotting. Error bars represent 1s confidence intervals estimated from biological replicates (N = 3 to N = 5) (left). Model fits and experimental data of Huh7.5-TetON-USP18 treated with doxycycline for 24 h and stimulated with 1,400 pM IFNa or parental Huh7.5 cells prestimulated with 0 or 1,400 pM IFNa and stimulated with 1,400 pM IFNa after 24 h are shown. The dynamics of IFNa-induced phosphorylation of STAT1 and STAT2 and induction of USP18 are depicted. Experimental data were obtained by quantitative immunoblotting using chemiluminescence and CCD camera-based device (ImageQuant). For modeling purposes, data from Huh7.5-TetON empty vector control and untransduced Huh7.5 are combined to one condition. Data are displayed as filled circles with errors representing 1s confidence intervals estimated from biological replicates (N = 3 to N = 4) using a combined scaling and error model. Line represents model trajectories.Scheme of possible mechanisms for SOCS1- and USP18-induced receptor degradation. Models with different structures concerning SOCS1- and USP18-catalyzed degradation of active receptor complexes (aRecIFN) were tested. Vertical lines denote separate enzymatic reactions, multiplication sign denotes cooperative enzymatic reactions. For each of the four different model structures, the resulting model trajectories of the best fit of pSTAT1 in the nucleus are exemplarily shown as dashed lines. Data are displayed as filled circles with errors representing 1s confidence intervals estimated from biological replicates (N = 1 to N = 38).Both SOCS1- and SOCS1:USP18-induced receptor degradation is required. Bayesian information criterion (BIC) was used to evaluate goodness-of-fit for the four different models shown in (C). The model “SOCS1 | SOCS1 × USP18” that shows the best performance comprises degradation of the active receptor complexes by both SOCS1 and a SOCS1:USP18 complex.

Fig 5: Snail1 could effectively reverse the influence of USP18 on cell proliferation, migration, invasion, and EMT of CRC cells. a, b Western blot and qRT-PCR analysis of the optimal interference sequence and overexpression sequence of Snail 1 siRNA. c, d CCK8 analysis of the USP18 knocked-down SW480 cells and USP18 over-expressed DLD1 cells with Snail1 siRNA and overexpression plasmid treatments. e, f Scratch experiment analysis of the USP18 over-expressed DLD1 cells and USP18 knocked-down SW480 cells with Snail1 siRNA and overexpression plasmid treatments. g, h Three common chemotherapeutics (fluorouracial, doxorubicin and cisplatin) for cell survival rate analysis of the USP18 knocked-down SW480 cells and USP18 over-expressed DLD1 cells with Snail1 siRNA and overexpression plasmids treatments. *P < 0.05, **P < 0.01, ***P < 0.001