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: Model calibration with IFNa-induced signal transduction in Huh7.5 upon prestimulation and stimulation with IFNaGrowth factor-depleted Huh7.5 cells were prestimulated with 2.8, 28, 1,400 pM IFNa or left untreated and were stimulated with 1,400 pM IFNa 24 h later. IFNa-induced signaling was measured by time-resolved quantitative immunoblotting and detected with chemiluminescence using a CCD camera-based device. Data were normalized to reference proteins Calnexin or HDAC1, scaled and subjected to model calibration. Model calibration with time-resolved IFNa-induced phosphorylation of STAT1 and STAT2 and induced feedback proteins upon prestimulation with 0, 2.8, 28, or 1,400 pM IFNa. Cytoplasmic lysates were subjected to quantitative immunoblotting. Experimental data were represented by filled circles with errors representing 1s confidence intervals estimated from biological replicates (N = 3 to N = 23) using a combined scaling and error model. Model trajectories are represented by lines. pSTAT1, pSTAT2 represent phosphorylated STAT1 and STAT2 on residue Tyr701 and Tyr690, respectively. tSTAT1 and tSTAT2 represent total form of STAT1 and STAT2 comprising both phosphorylated and unphosphorylated STAT1, STAT2, respectively.Model calibration with time-resolved IFNa-induced feedback transcripts upon prestimulation with 0, 2.8, 28, or 1,400 pM IFNa, assessed by qRT-PCR. mRNA levels were normalized to the geometric mean of reference genes GAPDH, HPRT, and TBP. Experimental data are represented by filled circles with errors representing 1s confidence intervals estimated from biological replicates (N = 3 to N = 14) using a combined scaling and error model. Model trajectories are represented by lines.Model calibration with the amount of molecules per cell of STAT1, STAT2, IRF9, and USP18 determined 24 h after prestimulation with 0, 2.8, 28, or 1,400 pM IFNa. Calibrator proteins were spiked into 10 µg of total protein lysate and subjected to quantitative immunoblotting. Immunoblot detection was performed by chemiluminescence using a CCD camera-based device. Averaged values (N = 4) are displayed with standard deviations. Green squares indicate amounts estimated by the mathematical model.Model calibration of IFNa-induced phosphorylation of STAT1 and STAT2 upon stimulation of Huh7.5 prestimulated with 0, 2.8, 28, or 1,400 pM IFNa. Nuclear lysates were subjected to quantitative immunoblotting. Experimental data are represented by filled circles with errors representing 1s confidence interval estimated from biological replicates (N = 4 to N = 22) using a combined scaling and error model. Model trajectories are represented by lines.

Fig 4: mRNA accumulation of indicated deubiquitinating enzymes in human tongue squamous carcinoma cells SCC9 and SCC25(A) Viral production of oHSV-1 T1012G in human tongue squamous carcinoma cells SCC9 and SCC25. (B) Constitutive accumulation of selected DUB mRNA in SCC9 and SCC25 cells. (C) Accumulation of USP18 and USP20 mRNA in oHSV-1 T1012G-infected SCC9 and SCC25 cells. Error bar, mean ± SEM.

Fig 5: IFN-a unresponsiveness depends on ISG15 and USP18 in cells that overexpress IFN-?4 or treated with IFN-?4. (A) PHHs with the IFNL4 ?G/?G genotype were infected with JFH1 HCVcc at 5 MOI. After 72 hours, the cells were treated with 100 IU/ml of IFN-a for 30 minutes. (B) Huh-7 cells were transfected with control or IFN-?4-expressing plasmids. After 72 hours, the cells were treated with 100 IU IFN-a for 15 minutes. The cells were harvested, and protein expression was analysed by immunoblotting. (C) Huh-7 cells were transfected with control, USP18-, or ISG15-targeting siRNAs. After 24 hours, the cells were transfected with control or IFN-?4-expressing plasmids. After 48 hours, the cells were re-transfected with the respective siRNAs. The cells were harvested, and gene expression was analysed by immunoblotting 24 hours after the second siRNA transfection. (D,E) Huh-7 cells were transfected with control, USP18-, or ISG15-targeting siRNAs. After 24 hours, the cells were transfected with control or IFN-?4-expressing plasmids. After 48 hours, the cells were re-transfected with the respective siRNAs. After 48 hours, the cells were treated with 100 IU/ml of IFN-a for 15 minutes (D) or for 6 hours (E). Then the cells were harvested, and protein and gene expression were analysed by immunoblotting (D) or real-time qPCR (E). All the data are representative of two independent experiments. (F) Huh-7 cells were treated with or without 100 ng/mL recombinant IFN-?4. After 48 hours, the cells were transfected with control siRNAs, USP18-, or ISG15-targeting siRNAs. The USP18-, or ISG15-targeting siRNAs used in this experiment had different sequences from siRNAs used in the experiment for (D). After 48 hours, the cells were treated with 100 IU/ml of IFN-a for 15 minutes. Then the cells were harvested, and protein expression were analysed by immunoblotting. (G) PHHs with IFNL4 ?G/?G genotype were infected with JFH1 HCVcc at 15 MOI. After 6 hours, the cells were treated with 10 µg/ml anti-IFN-?1 and 10 µg/ml anti-IFN-?2/3 or IgG for 72 hours. The cells were harvested, and ISG15 gene expression was analysed by real-time qPCR. Data are presented as means ± S.E.M. **P = 0.01, ***P = 0.001 (Student’s t-test).