Journal: bioRxiv

Article Title: Mechanisms of Differential Signal Transduction by IFNLR1 Variants

doi: 10.1101/2025.10.03.677101

Figure Lengend Snippet: (A) Cartoon depicting IFNL bound to each IFNLR1 variant and complexed with IL10RB with proximal kinases JAK1 and TYK2 engaged. Note the truncation in the Box1 and Box2 JAK1 binding motifs within IFNLR1 variant 2. (B) Alignment of the JAK1 binding motifs for IFNLR1 variant 1 (Q8IU57; top sequence) and IFNLR1 variant 2 (Q8IU57-2; bottom sequence). Box 1 (blue highlight) and Box 2 (yellow highlight) domains are indicated and reveal the 29 amino acid truncation within the cytoplasmic domain of IFNLR1 variant 2 that eliminates the C-terminal residues of Box 1 and all but two residues of Box 2. Created in BioRender. Novotny, L. (2025) https://BioRender.com/24aqcgc .

Article Snippet: Variant-expressing HEK293T cells and WT iHeps treated +/-dox (100ng/ml) and IFNLR1 -KO iHeps treated + dox (100ng/ml dox) for 22h were incubated in medium +/-0.01-1.0μM upadacitinib (a JAK1 inhibitor, Tocris) or 0.01-1.0μM deucravacitinib (a TYK2 inhibitor, TargetMol) for 2h prior to stimulation +/- IFNL3 (100ng/ml, 24h) in the continued presence of inhibitor.

Techniques: Variant Assay, Binding Assay, Sequencing

Journal: bioRxiv

Article Title: Mechanisms of Differential Signal Transduction by IFNLR1 Variants

doi: 10.1101/2025.10.03.677101

Figure Lengend Snippet: Western blot analysis of whole cell lysates from ( A ) WT iHeps and ( B ) IFNLR1 -KO iHeps +/-dox-induced for 24h then treated +/-IFNL3 or IFNA2 for 15min. GAPDH served as an indicator of equivalent protein loading per lane. The ratio of phosphorylated to total protein for ( C ) dox-uninduced WT iHeps and ( D ) dox-induced IFNLR1 -KO iHeps +/-IFNL3 is shown as a percentage, based on integrated band intensity determined in ImageJ.

Article Snippet: Variant-expressing HEK293T cells and WT iHeps treated +/-dox (100ng/ml) and IFNLR1 -KO iHeps treated + dox (100ng/ml dox) for 22h were incubated in medium +/-0.01-1.0μM upadacitinib (a JAK1 inhibitor, Tocris) or 0.01-1.0μM deucravacitinib (a TYK2 inhibitor, TargetMol) for 2h prior to stimulation +/- IFNL3 (100ng/ml, 24h) in the continued presence of inhibitor.

Techniques: Western Blot

Journal: bioRxiv

Article Title: Mechanisms of Differential Signal Transduction by IFNLR1 Variants

doi: 10.1101/2025.10.03.677101

Figure Lengend Snippet: ( A ) Model depicting the mechanisms of distinct signaling outcomes imparted by ternary complexes composed of IFNL3, IL10RB and either IFNLR1 variant 1 or 2. Greater arrow width indicates higher association and/or phosphorylation (JAK1, STAT1, STAT2), greater internalization of receptor complexes, or higher induction of gene expression. IFNLR1 variant 1 containing heterodimers are depicted to have greater stability of JAK1 and/or pJAK1 binding and to be more prone to internalization than IFNLR1 variant 2 containing heterodimers. This correlates with variant 1 mediating more efficient phosphorylation of STAT1 and STAT2 and supporting higher expression of antiviral ISGs and de novo expression of proinflammatory ISGs compared to variant 2. ( B ) Model depicting assemblage of multimeric clusters containing multiple heterodimers of IFNL3-bound IFNLR1 variants in complex with IL10RB. In this model, proximity of JAK1 molecules bound to the cytoplasmic domains of IFNLR1 variants 1 and 2 could participate in TYK2-independent transphosphorylation. The relative abundance of IFNLR1 variants within multimeric complexes would influence the nature of receptor internalization, STAT phosphorylation, and downstream gene expression. The TYK2 dependence of signaling differs for antiviral vs. proinflammatory ISG expression and has a complex relationship with the relative abundance of IFNLR1 variants. Soluble variant 3, not studied in these experiments, is included in the model for consideration. Created in BioRender. Novotny, L. (2025) https://BioRender.com/24aqcgc .

Article Snippet: Variant-expressing HEK293T cells and WT iHeps treated +/-dox (100ng/ml) and IFNLR1 -KO iHeps treated + dox (100ng/ml dox) for 22h were incubated in medium +/-0.01-1.0μM upadacitinib (a JAK1 inhibitor, Tocris) or 0.01-1.0μM deucravacitinib (a TYK2 inhibitor, TargetMol) for 2h prior to stimulation +/- IFNL3 (100ng/ml, 24h) in the continued presence of inhibitor.

Techniques: Variant Assay, Phospho-proteomics, Gene Expression, Binding Assay, Expressing

Journal: BMC Molecular and Cell Biology

Article Title: Minicircle DNA vector expressing interferon-lambda-3 inhibits hepatitis B virus replication and expression in hepatocyte-derived cell line

doi: 10.1186/s12860-020-00250-9

Figure Lengend Snippet: MC.IFNλ3 permits hepatocyte-specific expression of IFNλ3. HepG2.2.15, HEK293 and Hela cells were transfected with MC vectors. a Schematic illustration of the MC.IFNs. MC.IFNα is 1656-bp in length, MC.IFNλ3 is 1677-bp in length. attR represents a 36-bp attR recombinant site. ApoE indicates ApoE promoter. CDS represents coding sequence. bpA represents bovine growth hormone polyadenylation signal. b The expression of IFNα and IFNλ3 in cell lysate was determined by Western Blot at 3 days post-transfection. Lane 1–5 represents the untreated control (HepG2.2.15 cells without MC transfection), MC.IFNα transfected HepG2.2.15 cells, and MC.IFNλ3 transfected HepG2.2.15 cells, MC.IFNλ3 transfected HEK293 cells, MC.IFNλ3 transfected Hela cells, respectively

Article Snippet: After blocked the non-specific binding sites with 5% skim milk in TBST (Sigma, US), the membrane was subjected to immunoblotting using a primary antibody listed as below: the rabbit polyclonal antibody specific to IFN⍺ (ProteinTech, US; #18013–1-AP) and IFNλ3 (ProteinTech, US; #24199–1-AP); the rabbit monoclonal antibodies specific to JAK1 (Cell Signaling Technology, US; #3344) and phosphorylated JAK1 (p-JAK1) (Cell Signaling Technology, US; #3331); the rabbit polyclonal or monoclonal antibodies specific to STAT1 (Abcam, UK; #ab2415), STAT2 (Abcam, UK; #ab53149), phosphorylated STAT1 (p-STAT1) (Cell Signaling Technology, US; #9171) and phosphorylated STAT2 (p-STAT2) (Millipore, US; #07–224).

Techniques: Expressing, Transfection, Recombinant, Sequencing, Western Blot, Control

Journal: BMC Molecular and Cell Biology

Article Title: Minicircle DNA vector expressing interferon-lambda-3 inhibits hepatitis B virus replication and expression in hepatocyte-derived cell line

doi: 10.1186/s12860-020-00250-9

Figure Lengend Snippet: MC.IFNλ3 inhibits viral antigens expression and viral DNA replication in HepG2.2.15 cells. HepG2.2.15 cells were transfected with MC.IFNλ3 and MC.IFNα. While the untreated HepG2.2.15 cells served as a blank control (Blank). The levels of viral antigens, namely HBsAg ( a ) and HBeAg ( b ), and viral DNA in cell culture supernatant were determined by chemiluminiscence and qPCR, respectively, at the indicated time-points (3 or 6 days post-transfection). All data are shown as mean ± SD from three independent experiments. * indicates statistically significant ( P -value < 0.05), ns indicates not significant ( P -value > 0.05)

Article Snippet: After blocked the non-specific binding sites with 5% skim milk in TBST (Sigma, US), the membrane was subjected to immunoblotting using a primary antibody listed as below: the rabbit polyclonal antibody specific to IFN⍺ (ProteinTech, US; #18013–1-AP) and IFNλ3 (ProteinTech, US; #24199–1-AP); the rabbit monoclonal antibodies specific to JAK1 (Cell Signaling Technology, US; #3344) and phosphorylated JAK1 (p-JAK1) (Cell Signaling Technology, US; #3331); the rabbit polyclonal or monoclonal antibodies specific to STAT1 (Abcam, UK; #ab2415), STAT2 (Abcam, UK; #ab53149), phosphorylated STAT1 (p-STAT1) (Cell Signaling Technology, US; #9171) and phosphorylated STAT2 (p-STAT2) (Millipore, US; #07–224).

Techniques: Expressing, Transfection, Control, Cell Culture

Journal: BMC Molecular and Cell Biology

Article Title: Minicircle DNA vector expressing interferon-lambda-3 inhibits hepatitis B virus replication and expression in hepatocyte-derived cell line

doi: 10.1186/s12860-020-00250-9

Figure Lengend Snippet: Viral antigens and viral DNA in HepG2.2.15 cell culture supernatant after transfection

Article Snippet: After blocked the non-specific binding sites with 5% skim milk in TBST (Sigma, US), the membrane was subjected to immunoblotting using a primary antibody listed as below: the rabbit polyclonal antibody specific to IFN⍺ (ProteinTech, US; #18013–1-AP) and IFNλ3 (ProteinTech, US; #24199–1-AP); the rabbit monoclonal antibodies specific to JAK1 (Cell Signaling Technology, US; #3344) and phosphorylated JAK1 (p-JAK1) (Cell Signaling Technology, US; #3331); the rabbit polyclonal or monoclonal antibodies specific to STAT1 (Abcam, UK; #ab2415), STAT2 (Abcam, UK; #ab53149), phosphorylated STAT1 (p-STAT1) (Cell Signaling Technology, US; #9171) and phosphorylated STAT2 (p-STAT2) (Millipore, US; #07–224).

Techniques: Cell Culture, Transfection, Control

Journal: BMC Molecular and Cell Biology

Article Title: Minicircle DNA vector expressing interferon-lambda-3 inhibits hepatitis B virus replication and expression in hepatocyte-derived cell line

doi: 10.1186/s12860-020-00250-9

Figure Lengend Snippet: MC.IFNλ3 induce JAK1 and STAT1/STAT2 phosphorylation in HepG2.2.15 cells. HepG2.2.15 cells were transfected with MC vectors. The levels of a STAT1/STAT2 proteins and their phosphorylated form (p-STAT1/p-STAT2), b JAK1 and phosphorylated JAK1 (p-JAK1) in transfected HepG2.2.15 cells were determined by Western Blot at 6 days post-transfection. Lane 1, 2 and 3 represents untreated Control, MC.IFNα, and MC.IFNλ3 group, respectively

Article Snippet: After blocked the non-specific binding sites with 5% skim milk in TBST (Sigma, US), the membrane was subjected to immunoblotting using a primary antibody listed as below: the rabbit polyclonal antibody specific to IFN⍺ (ProteinTech, US; #18013–1-AP) and IFNλ3 (ProteinTech, US; #24199–1-AP); the rabbit monoclonal antibodies specific to JAK1 (Cell Signaling Technology, US; #3344) and phosphorylated JAK1 (p-JAK1) (Cell Signaling Technology, US; #3331); the rabbit polyclonal or monoclonal antibodies specific to STAT1 (Abcam, UK; #ab2415), STAT2 (Abcam, UK; #ab53149), phosphorylated STAT1 (p-STAT1) (Cell Signaling Technology, US; #9171) and phosphorylated STAT2 (p-STAT2) (Millipore, US; #07–224).

Techniques: Phospho-proteomics, Transfection, Western Blot, Control

Journal: BMC Molecular and Cell Biology

Article Title: Minicircle DNA vector expressing interferon-lambda-3 inhibits hepatitis B virus replication and expression in hepatocyte-derived cell line

doi: 10.1186/s12860-020-00250-9

Figure Lengend Snippet: MC.IFNλ3 up-regulates ISGs expression in HepG2.2.15 cells. MC.IFNλ3 up-regulates ISGs expression in HepG2.2.15 cells. The relative mRNA transcriptional levels of ten ISGs MC transfected HepG2.2.15 cells were quantified at 3 or 6 days post-transfection by qPCR. The ISGs mRNA levels in HepG2.2.15 cells after MC.IFNλ3 ( a ) and MC.IFNα ( b ) treatment were compared between 3 days and 6 days post-transfection groups. The ISGs mRNA levels in HepG2.2.15 cells between MC.IFNλ3 and MC.IFNα treatment groups were compared at 3 days ( c ) or 6 days ( d ) post-transfection. All data are shown as mean ± SD from three independent experiments

Article Snippet: After blocked the non-specific binding sites with 5% skim milk in TBST (Sigma, US), the membrane was subjected to immunoblotting using a primary antibody listed as below: the rabbit polyclonal antibody specific to IFN⍺ (ProteinTech, US; #18013–1-AP) and IFNλ3 (ProteinTech, US; #24199–1-AP); the rabbit monoclonal antibodies specific to JAK1 (Cell Signaling Technology, US; #3344) and phosphorylated JAK1 (p-JAK1) (Cell Signaling Technology, US; #3331); the rabbit polyclonal or monoclonal antibodies specific to STAT1 (Abcam, UK; #ab2415), STAT2 (Abcam, UK; #ab53149), phosphorylated STAT1 (p-STAT1) (Cell Signaling Technology, US; #9171) and phosphorylated STAT2 (p-STAT2) (Millipore, US; #07–224).

Techniques: Expressing, Transfection