Journal: Nature Communications

Article Title: Zinc is a potent and specific inhibitor of IFN-λ3 signalling

doi: 10.1038/ncomms15245

Figure Lengend Snippet: ( a ) Huh-7 cells were treated with 50 U ml −1 IFN-α or 100 ng ml −1 IFN-λ3 in combination with increasing amounts of zinc (0, 5 and 50 μM ZnSO 4 ) or 5 μM ZnSO 4 in combination with 10 mg ml −1 or 50 mg ml −1 BSA to sequester zinc. STAT1 phosphorylation 30 min post IFN-λ3 treatment was dose-dependently inhibited by ZnSO 4 , and reversed with albumin. IFN-α treatment however, was unaffected. ( b ) The expression of IFN-λ3-induced ISGs, MX1 and viperin , were affected in a similar manner, with minimal effects on IFN-α-mediated ISG expression (Welch's t -test). ( c ) To determine if zinc inhibits the antiviral response, Huh-7 cells infected with the JFH-1 strain of HCV and H1N1 influenza virus were treated with IFN-α and IFN-λ3 in combination with zinc. IFN-α-reduced JFH-1 intracellular RNA by ∼75% independent of zinc concentration, however the antiviral activity of IFN-λ3 was significantly reduced from ∼50% to ∼20% with 50 μM ZnSO 4 . The antiviral activity of IFN-α against influenza virus was only partially inhibited by zinc, however a similar dose-dependent inhibition of IFN-λ3 antiviral activity was observed, almost ablating its effects at 50 μM ZnSO 4 (Welch's t -test). Data are representative of two (western blotting) and three (gene expression) independent experiments. * P <0.05, ** P <0.01, *** P <0.001, (mean±s.e.). UT, untreated.

Article Snippet: Protein was transferred on to nitrocellulose membrane, and β-actin and STAT1 were probed using STAT1 (Santa Cruz Biotechnology, SC-345 clone C-24, rabbit anti-human, 1:2,000 dilution), Tyr701 p-STAT1 (Cell Signalling, 9167 clone 58D6, rabbit anti-human, 1:500 dilution) and β-actin (Sigma-Aldrich, A1978 clone AC-15, mouse anti-human, 1:10,000 dilution) antibodies.

Techniques: Phospho-proteomics, Expressing, Infection, Virus, Concentration Assay, Activity Assay, Inhibition, Western Blot, Gene Expression

Journal: Nature Communications

Article Title: Zinc is a potent and specific inhibitor of IFN-λ3 signalling

doi: 10.1038/ncomms15245

Figure Lengend Snippet: Zinc-regulated transcriptome disease class and biological process as identified by DAVID functional annotation.

Article Snippet: Protein was transferred on to nitrocellulose membrane, and β-actin and STAT1 were probed using STAT1 (Santa Cruz Biotechnology, SC-345 clone C-24, rabbit anti-human, 1:2,000 dilution), Tyr701 p-STAT1 (Cell Signalling, 9167 clone 58D6, rabbit anti-human, 1:500 dilution) and β-actin (Sigma-Aldrich, A1978 clone AC-15, mouse anti-human, 1:10,000 dilution) antibodies.

Techniques: Functional Assay, Infection, Transduction

Journal: Nature Communications

Article Title: Zinc is a potent and specific inhibitor of IFN-λ3 signalling

doi: 10.1038/ncomms15245

Figure Lengend Snippet: ( a ) To ascertain the cellular location of zinc inhibition following ZnSO 4 treatment, Huh-7 cells were pre-treated for 24 h with 50 μM ZnSO 4 , washed thoroughly, and then treated with IFN-λ3 ±50 μM ZnSO 4 for 15 min. STAT1 phosphorylation was significantly inhibited only when IFN-λ3 was administered in the presence of zinc, suggesting that zinc inhibits IFN-λ3 signalling at the receptor. ( b ) To identify cytokine zinc binding, 200 ng of IFN-α or IFN-λ3 were added to a zinc loaded IMAC-affinity resin for 2 h, after which bound protein was examined by SDS–PAGE. Dimeric forms of IFN-α and IFN-λ3 bound the zinc resin, as did monomeric IFN-λ3. ( c ) Using protein G beads, Co-IP was performed on Huh-7 cell lysates using recombinant IFN-λ3 alone or with the addition of 50 μM ZnSO 4 . Co-IP of both IFN-λ3 (66% reduction) and IFNLR1 (40% reduction) was reduced in the presence of zinc, when IFNLR1 and IFN-λ3 were immunoprecipitated respectively, demonstrating a direct inhibition of receptor:cytokine interaction by zinc. ( d ) Co-IP studies were confirmed using proximity ligation assays to examine the interaction of IFN-λ3 and IFNLR1 in the presence of zinc in situ . Confocal microscopy of IFN-λ3 treated Huh-7 cells demonstrated a reduction in fluorescent signals (red fluorescent spots), indicative of IFNLR1:IFN-λ3 interaction (within 40 nm). No signal was observed within the negative control assay lacking the primary IFN-λ3 antibody (1° Ab) or lacking the addition of IFN-λ3. ( e ) Proximity ligation signals per cell were quantified using ImageJ software, demonstrating a 50% decrease in IFNLR1:IFN-λ3 interactions per cell (Mann–Whitney test). ( d ) Scale bars, 10 μm. Data are representative of two independent experiments, *** P <0.001, (mean±s.e.).

Article Snippet: Protein was transferred on to nitrocellulose membrane, and β-actin and STAT1 were probed using STAT1 (Santa Cruz Biotechnology, SC-345 clone C-24, rabbit anti-human, 1:2,000 dilution), Tyr701 p-STAT1 (Cell Signalling, 9167 clone 58D6, rabbit anti-human, 1:500 dilution) and β-actin (Sigma-Aldrich, A1978 clone AC-15, mouse anti-human, 1:10,000 dilution) antibodies.

Techniques: Inhibition, Phospho-proteomics, Binding Assay, SDS Page, Co-Immunoprecipitation Assay, Recombinant, Immunoprecipitation, Ligation, In Situ, Confocal Microscopy, Negative Control, Software, MANN-WHITNEY