Journal: bioRxiv

Article Title: IFN-λ4 may contribute to HCV persistence by increasing ER stress and enhancing IRF1 signaling

doi: 10.1101/2020.10.28.359398

Figure Lengend Snippet: Differentially expressed genes (DEGs, P-FDR < 0.05) were identified by RNA-seq analysis in IFN-λ3-GFP, IFN-λ4-GFP and IFN-λ4-GFP-IFNLR1 KO HepG2 cells after 72 hrs of induction by dox, comparing to controls (dox-conditions). Cutoff threshold (fold change > +/-1.5) is indicated by dotted lines. (A) Analysis of all DEGs (n=3251) detected for IFN-λ4-GFP or IFN-λ4-GFP-IFNLR1 KO cells. In blue - DEGs (n=2,735) specific to IFN-λ4-GFP and considered IFNLR1-dependent. In black - DEGs (n=145) shared between both groups and considered IFNLR1-independent. In orange - DEGs (n=371) specific to IFN-λ4-GFP-IFNLR1 KO . (B) DEGs of IFN-λ4-GFP analyzed in IFN-λ3-GFP transcriptome. In black - DEGs (n=1,506) shared in IFN-λ4-GFP and IFN-λ3-GFP and in blue - IFN-λ4-signature DEGs (n=1,229) detected in IFN-λ4-GFP but not in IFN-λ3-GFP producing cells. Additional details are provided in Fig. S3 and Table S6. (C , D) Cell cycle analysis of cells synchronized by 24 hrs of serum starvation, treated with or without dox (0.5 µg/ml) for 72 hrs and analyzed by flow cytometry after PI staining. The plot shows a representative picture and the percentage of cells in each phase of the cell cycle. All data are shown as mean± SEM from triplicate experiments. *, P < 0.05. (E , F) Bromodeoxyuridine (BRDU, %) incorporation indicating cell proliferation in HepG2 cells expressing IFN-λ3-GFP, IFN-λ4-GFP and IFN-λ4-GFP-IFNLR1KO. Cells were cocultured with HepG2 cells labeled with Far Red proliferation dye, dox-induced for 72 hrs and treated with BRDU for 3 hrs before analysis. Gates show HepG2 cells exposed to IFN-λs (IFN-λ treated cells) and HepG2 expressing IFN-λs. P-values compare corresponding dox+ vs. dox-HepG2 cells, ** p<0.01, Student’s T-test. Graphs represent one of three independent experiments, each in biological triplicates.

Article Snippet: The membranes were probed with primary antibodies against IFNLR1 (#NBP1-84381, Novus Biologicals), STAT1 (#9172, Cell Signaling Technology), phospho-STAT1 (Tyr701, #58D6, Cell Signaling Technology), IFN-λ4 (ab196984; Abcam), GAPDH (ab37168, Abcam) and HRP-linked secondary antibody, goat anti-rabbit IgG (#7074; Cell Signaling Technology) or goat anti-mouse IgG (San Cruz, sc-2031).

Techniques: RNA Sequencing, Cell Cycle Assay, Flow Cytometry, Staining, BrdU Incorporation Assay, Expressing, Labeling

Journal: bioRxiv

Article Title: IFN-λ4 may contribute to HCV persistence by increasing ER stress and enhancing IRF1 signaling

doi: 10.1101/2020.10.28.359398

Figure Lengend Snippet: (A) Unsupervised clustering of activities of 54 regulons that were significantly and directionally enriched with HepG2-DEGs in the set of 885 of TCGA-LIHC regulons. The heatmap shows differences in activity scores (dES) for the IFN-λ4-enriched regulons organized by GSEA-2T results for the IFN-λ4-GFP, IFN-λ3-GFP and IFN-λ4-GFP-IFNLR1 KO DEG signatures. Cluster I: regulons with dES > 0 in IFN-λ4-GFP; cluster II: regulons with dES < 0 in IFN-λ4-GFP. Table S7 provides the regulon activity scores presented in Figure 2A. (B-G) GSEA-2T plots for IRF1 and IRF2, respectively, in DEG signatures for (B-C) IFN-λ3-GFP, ( D-E ) IFN-λ4-GFP, and ( F-G ) IFN-λ4-GFP-IFNLR1 KO . (H) Inhibition of proliferation in IFN-λ4-GFP HepG2 cells, for one of three independent experiments. Cells were treated with IRF1 siRNA for 24 hrs, labeled with Far Red proliferation dye and dox-induced at indicated concentrations for 72 hrs. Proliferation was assessed by flow cytometry with a graph representing the geometric mean expression of Far Red proliferation dye with higher values indicating reduced cell proliferation. P-values compare dox-treated control siRNA with dox-treated IRF1 siRNA. ** p<0.01, Student’s T-test. Below: Western blots showing IRF1 protein levels following siRNA knockdown.

Article Snippet: The membranes were probed with primary antibodies against IFNLR1 (#NBP1-84381, Novus Biologicals), STAT1 (#9172, Cell Signaling Technology), phospho-STAT1 (Tyr701, #58D6, Cell Signaling Technology), IFN-λ4 (ab196984; Abcam), GAPDH (ab37168, Abcam) and HRP-linked secondary antibody, goat anti-rabbit IgG (#7074; Cell Signaling Technology) or goat anti-mouse IgG (San Cruz, sc-2031).

Techniques: Activity Assay, Inhibition, Labeling, Flow Cytometry, Expressing, Control, Western Blot, Knockdown

Journal: bioRxiv

Article Title: IFN-λ4 may contribute to HCV persistence by increasing ER stress and enhancing IRF1 signaling

doi: 10.1101/2020.10.28.359398

Figure Lengend Snippet: Representative confocal images of HepG2 cells transduced with a mammalian baculovirus delivery system (BacMam) of GFP-tagged proteins targeting specific organelles - lysosomes, Golgi, early and late endosomes. After transduction for 6 hrs, cells were transiently transfected with Halo-tagged constructs for IFN-λ4 or control for indicated times, stained with cell-permeant Halo-tag ligand TMR (red), and imaged. (A) Confocal images showing IFN-λ4 accumulation in lysosomes but not in early endosomes. (B) Late endosomal trafficking of IFN-λ4, with the inset showing larger magnification. (C) Unfolded protein response (UPR) is represented by lysosomal enlargement after protein accumulation. (D) Live images of IFN-λ4-expressing HepG2 cells undergoing apoptosis, characterized by membrane blebbing and cell death. Images were scanned every minute for 12 hrs. Scale bars – 10 um. (E) Apoptosis detection with ApoTox-Glo assays in corresponding untreated and dox-induced cells for indicated time points. RLU, relative luminescence units. (F) Graph showing counts from colony formation assay for HepG2 cells expressing IFN-λ4 or IFNLR1 KO grown in 6-well plates with or without dox for 13 days. Cell colonies were stained with crystal violet and manually counted. The graph represents the number of colonies as a percentage of initial plated counts. (G,H) mRNA (G) and protein levels ( H ) of DDIT3 after siRNA knockdown tested by qRT-PCR and Western blot assays, respectively. (I-J) Apoptosis (I) and cell viability (J) assays were performed after siRNA knockdown of DDIT3 in dox-induced IFN-λ4-GFP cells. * p<0.05, ** p< 0.01, *** p<0.001.

Article Snippet: The membranes were probed with primary antibodies against IFNLR1 (#NBP1-84381, Novus Biologicals), STAT1 (#9172, Cell Signaling Technology), phospho-STAT1 (Tyr701, #58D6, Cell Signaling Technology), IFN-λ4 (ab196984; Abcam), GAPDH (ab37168, Abcam) and HRP-linked secondary antibody, goat anti-rabbit IgG (#7074; Cell Signaling Technology) or goat anti-mouse IgG (San Cruz, sc-2031).

Techniques: Transduction, Transfection, Construct, Control, Staining, Expressing, Membrane, Colony Assay, Knockdown, Quantitative RT-PCR, Western Blot

Journal: Immunity

Article Title: Human TLR-7-, -8-, and -9-Mediated Induction of IFN-α/β and -λ Is IRAK-4 Dependent and Redundant for Protective Immunity to Viruses

doi: 10.1016/j.immuni.2005.09.016

Figure Lengend Snippet: Production of IFN-α/β, IFN-λ, and TNF-α after Stimulation with Viruses in IRAK-4-Deficient Blood Cells PBMCs from controls and/or from IRAK-4-deficient patients P2, P3, or P7 were left unstimulated or stimulated with various intact and UV-inactivated viruses. (A) IFN-β mRNA levels were analyzed by reverse transcription and real-time quantitative PCR 24 hr after stimulation with intact viruses (see for the multiplicities of infection, moi). Means and standard deviations (SD) were calculated for the controls from three independent individuals, each tested once. The patients were each tested once. (B and C) IFN-α secretion (B) and IFN-λ secretion (C) were measured by ELISA after 24 hr of stimulation. Means and standard deviations (SD) were calculated for the controls from 14 independent individuals, each tested once. (D) IFN-α secretion by control PBMCs was measured by ELISA after 24 hr of stimulation by intact or UV-inactivated viruses. Means and standard deviations (SD) were calculated for the controls from seven independent individuals, each tested once. (E) TNF-α secretion was measured by ELISA after 24 hr of stimulation by two intact viruses. Means and standard deviations (SD) for controls were calculated from two independent individuals.

Article Snippet: Plates were coated with 1 μg/ml of anti-human IFN-λ mAb (AF1598, R&D Systems) overnight at 4°C, and the IFN-λ levels in the supernatant were estimated with a secondary biotinylated anti-human IFN-λ mAb (BAF1598, R&D Systems) used at a concentration of 400 μg/ml ( ).

Techniques: Reverse Transcription, Real-time Polymerase Chain Reaction, Infection, Enzyme-linked Immunosorbent Assay, Control

Journal: Immunity

Article Title: Human TLR-7-, -8-, and -9-Mediated Induction of IFN-α/β and -λ Is IRAK-4 Dependent and Redundant for Protective Immunity to Viruses

doi: 10.1016/j.immuni.2005.09.016

Figure Lengend Snippet: IFN-β, IFN-λ, and IL-6 Induction in IRAK-4-Deficient Fibroblasts upon Stimulation by Poly(I:C), IL-1β, TNF-α, and Viruses Induction of IFN-β, IFN-λ, and IL-6 in response to poly(I:C) (50 μg/ml), IL-1β (20 ng/ml), TNF-α (10 ng/ml), and viral stimulations in IRAK-4-deficient (P1, P2, or P4) and control fibroblasts. (A and B) IFN-β mRNA induction, as determined by Q-PCR 2 hr after stimulation (A) and IFN-β, as determined by ELISA 24 hr after stimulation (B). Means and standard deviations (SD) were calculated for each patient from two experiments, and for the controls from two independent individuals, each tested twice. (C) IFN-β and IFN-λ as determined by ELISA 24 hr after stimulation by poly(I:C), following prior treatment with IFN-α2b (10 5 U/ml for 12 hr). This experiment is representative of two independent experiments. (D) IL-6, as determined by ELISA 24 hr after stimulation. Means and standard deviations (SD) were calculated for each patient from two experiments, and for the controls from two independent individuals, each tested twice. (E) IFN-β mRNA induction 24 hr after stimulation by seven intact viruses (see for the multiplicities of infection, moi). Means and standard deviations (SD) were calculated for each patient from two experiments, and for the controls from two independent individuals, each tested twice.

Article Snippet: Plates were coated with 1 μg/ml of anti-human IFN-λ mAb (AF1598, R&D Systems) overnight at 4°C, and the IFN-λ levels in the supernatant were estimated with a secondary biotinylated anti-human IFN-λ mAb (BAF1598, R&D Systems) used at a concentration of 400 μg/ml ( ).

Techniques: Control, Enzyme-linked Immunosorbent Assay, Infection