Journal:

Article Title: RPA is an initiation factor for human chromosomal DNA replication

doi:

Figure Lengend Snippet: RPA is recruited to DNA replication foci and becomes phosphorylated in vitro. (A) Comparison of bound RPA in G1 and S phase nuclei in vivo. Nuclear extracts (25 µg protein per lane) from mimosine- arrested late G1 phase and from early S phase cells were analysed by western blot using antibodies specific for MCM5 as a control and polyclonal antibody pAb-RPA1. (B) Nuclear binding and phosphorylation of RPA during initiation of DNA replication in vitro. G1 phase nuclei isolated from in vitro incubations done in replication buffer (lane 1), 100 µg S100 cytosolic extract (lane 2), 30 and 100 ng rhRPA (lanes 3 and 4) and 100 ng rhRPA plus 35 µg of fraction QB (lane 5). The samples shown in the right-hand panel were treated with λ phosphatase before loading onto the same gel. The Rpa70 and Rpa32 subunits were visualised with pAb-RPA1. Phosphorylated Rpa32 is indicated as pRpa32. (C) Confocal microscopy. G1 phase nuclei were incubated in buffer (top row), in 100 ng of rhRPA (second row), in 100 ng rhRPA supplemented with 35 µg QB (third row) and in 100 µg of unfractionated S100 (bottom row). High-resolution micrographs are shown of RPA foci (stained with pAb-RPA1, red), replication foci (dig-UTP, green) and merged images of the same set of nuclei.

Article Snippet: Phosphatase treatments were performed after the PBS wash by resuspending the nuclei in 20 µl of λ phosphatase buffer containing 2 mM MnCl 2 and incubating for 30 min at 30°C in the presence of 200 U λ phosphatase (NEB).

Techniques: In Vitro, In Vivo, Western Blot, Binding Assay, Isolation, Confocal Microscopy, Incubation, Staining

Journal: Tissue antigens

Article Title: Direct binding to antigen-coated beads refines the specificity and cross-reactivity of four monoclonal antibodies that recognize polymorphic epitopes of HLA class I molecules

doi: 10.1111/tan.12095

Figure Lengend Snippet: (A) HLA class I allotypes represented by the One Lambda Labscreen and Gen-Probe LifeCodes beadsets. (B) Binding of the monomorphic HLA class I antibody W6/32 to beads from One Lambda LabScreen (grey bars) and Gen-Probe LifeCodes (orange bars). The allotypes shown are those common to both beadsets.

Article Snippet: The specific reference as listed in the current publication is noted to the right. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 2 caption a7 (A) HLA class I allotypes represented by the One Lambda Labscreen and Gen-Probe LifeCodes beadsets. (B) Binding of the monomorphic HLA class I antibody W6/32 to beads from One Lambda LabScreen (grey bars) and Gen-Probe LifeCodes (orange bars).

Techniques: Binding Assay

Journal: Tissue antigens

Article Title: Direct binding to antigen-coated beads refines the specificity and cross-reactivity of four monoclonal antibodies that recognize polymorphic epitopes of HLA class I molecules

doi: 10.1111/tan.12095

Figure Lengend Snippet: (A) Binding of MA2.1 (1μg/ml) to beads coated with HLA class I allotypes from the One Lambda LabScreen (left panel) and Gen-Probe LifeCodes (right panel) beadsets. (B) Alignment of HLA class I allotypes showing selected residues in the α1 and α2 domains. Residues from allotypes that form the epitope recognized by MA2.1 are shaded in grey. (C) Space-filling model of the binding surface of HLA-A*02 (grey) with associated peptide (cyan). Residues highlighted in yellow fall within the footprint recognized by MA2.1. Residues 62–65 are critical for formation of the epitope recognized by MA2.1 and are highlighted in red.

Article Snippet: The specific reference as listed in the current publication is noted to the right. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 2 caption a7 (A) HLA class I allotypes represented by the One Lambda Labscreen and Gen-Probe LifeCodes beadsets. (B) Binding of the monomorphic HLA class I antibody W6/32 to beads from One Lambda LabScreen (grey bars) and Gen-Probe LifeCodes (orange bars).

Techniques: Binding Assay

Journal: Tissue antigens

Article Title: Direct binding to antigen-coated beads refines the specificity and cross-reactivity of four monoclonal antibodies that recognize polymorphic epitopes of HLA class I molecules

doi: 10.1111/tan.12095

Figure Lengend Snippet: (A) Binding of PA2.1 (1μg/ml) and BB7.2 (1μg/ml) to beads coated with HLA class I allotypes from the One Lambda LabScreen (left panel) and Gen-Probe LifeCodes (right panel) beadsets. (B) Binding of PA2.1 (50μg/ml) and BB7.2 (50μg/ml) to beads coated with HLA class I allotypes from the One Lambda LabScreen (left panel) and Gen-Probe LifeCodes (right panel) beadsets. (C) Alignment of HLA class I allotypes showing selected residues in the α2 domain. Residues from allotypes that form the epitope recognized by PA2.1 and BB7.2 are shaded in grey. (D) Space-filling model of HLA-A*02 (grey) with associated peptide (cyan). Residues highlighted in yellow fall within the footprint recognized by PA2.1 and BB7.2. Tryptophan at position 107 is considered critical for formation of the epitope recognized by PA2.1 and BB7.2 and is highlighted in red.

Article Snippet: The specific reference as listed in the current publication is noted to the right. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 2 caption a7 (A) HLA class I allotypes represented by the One Lambda Labscreen and Gen-Probe LifeCodes beadsets. (B) Binding of the monomorphic HLA class I antibody W6/32 to beads from One Lambda LabScreen (grey bars) and Gen-Probe LifeCodes (orange bars).

Techniques: Binding Assay

Journal: Tissue antigens

Article Title: Direct binding to antigen-coated beads refines the specificity and cross-reactivity of four monoclonal antibodies that recognize polymorphic epitopes of HLA class I molecules

doi: 10.1111/tan.12095

Figure Lengend Snippet: (A) Binding of BB7.1 (1μg/ml) to beads coated with HLA class I allotypes from the One Lambda LabScreen (left panel) and Gen-Probe LifeCodes (right panel) beadsets. (B) Alignment of HLA class I allotypes showing selected residues in the α1 and α2 domains. Residues from allotypes that form the epitope recognized by BB7.1 are shaded in grey. (C) Space-filling model of the binding surface of HLA-B*07 (grey) with associated peptide (cyan). Residues highlighted in yellow fall within the footprint recognized by BB7.1. Residues 63–71 in the a1 domain and position 131 in the α2 domain are critical for formation of the epitope recognized by BB7.1 and are highlighted in red.

Article Snippet: The specific reference as listed in the current publication is noted to the right. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 2 caption a7 (A) HLA class I allotypes represented by the One Lambda Labscreen and Gen-Probe LifeCodes beadsets. (B) Binding of the monomorphic HLA class I antibody W6/32 to beads from One Lambda LabScreen (grey bars) and Gen-Probe LifeCodes (orange bars).

Techniques: Binding Assay

Journal: PLOS Pathogens

Article Title: Basal IFNλ2/3 signaling is required for ISG expression and viral control in human intestinal epithelial cells

doi: 10.1371/journal.ppat.1013857

Figure Lengend Snippet: (A–D) T84 cells were seeded in 48-well plates and infected two days later with (A) VSV-GFP at an MOI of 1 for 7 hours, (B) MRV at an MOI of 1 for 16 hours, (C) RV-UnaG at an MOI of 1 for 16 hours and (D) VV-GFP at an MOI of 1 for 16 hours. (A) VSV-GFP (C) RV-UnaG and (D) VV-GFP infection was evaluated using live-cell microscopy; nuclei were stained with Hoechst. (B) MRV infection was assessed by immunostaining against the MRV µNS protein, and nuclei was stained using DAPI. (A–D) Representative fluorescence images showing virus (green) and nuclei (blue). Scale bar = 100 μm. (E–H) Total RNA was extracted from mock-infected or virus-infected T84 cells at (E) 7hpi of VSV-GFP and at 16hpi of (F) MRV, (G) RV-UnaG and (H) VV-GFP, followed by qRT-PCR analysis of IFNλ1 and IFNλ2/3 expression. Gene expression levels were normalized to TBP. (I–L) Supernatants collected from infected T84 cells at (I) 7hpi of VSV-GFP and at 16hpi of (J) MRV, (K) RV-UnaG and (L) VV-GFP, were analyzed by ELISA to quantify secreted IFNλ1 and IFNλ2/3 proteins following infection. Data represent ≥3 independent biological replicates. Statistical significance was determined by unpaired t-test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Error bars represent standard deviation with the mean as the center.

Article Snippet: For chronic supplementation with IFNλ2 and/or IFNλ3, IFNλ2/3 KO cells were seeded in the presence of 5 ng/mL IFNλ2 (R&D Systems #1587IL025/CF) and 1 ng/mL IFNλ3 (R&D Systems #5259-IL-025/CF).

Techniques: Infection, Microscopy, Staining, Immunostaining, Fluorescence, Virus, Quantitative RT-PCR, Expressing, Gene Expression, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: PLOS Pathogens

Article Title: Basal IFNλ2/3 signaling is required for ISG expression and viral control in human intestinal epithelial cells

doi: 10.1371/journal.ppat.1013857

Figure Lengend Snippet: T84 cells were seeded in 96-well plates and treated the following day with increasing concentrations (0.0001–300 ng/mL) of recombinant IFNλ1, IFNλ2, or IFNλ3 for 24 hours prior to infection. Cells were then infected with (A) VSV-Luc, (B) MRV, (C) RV-UnaG, or (D) VV-GFP, each at a multiplicity of infection (MOI) of 1. Infections were maintained in the presence of indicated dose of recombinant IFNλ1, IFNλ2, or IFNλ3. Infections were analyzed 7 hours post-infection (hpi) for VSV-Luc and 16 hpi for MRV, RV-UnaG, and VV-GFP. (A) VSV-Luc infection was quantified by luciferase assay. (B) MRV infection was assessed by immunofluorescence staining against the μNS protein, with DAPI used for nuclear staining. (C, D) RV-UnaG and VV-GFP infections were monitored via live-cell imaging; nuclei were stained with Hoechst. Data represent ≥3 independent biological replicates. Statistical significance between IFNλ-treated conditions and the untreated control (0 ng/ml) was determined using two-way ANOVA with Sidak’s post hoc correction (*P < 0.05, **P < 0.01, ***P < 0.001). Color-coded significance markers indicate comparisons between different doses and 0 ng/mL for each IFNλ subtype (IFNλ = blue, IFNλ2 = green and IFNλ3 = red). If not specified, comparisons are not significant (ns). Error bars represent standard deviation with the mean as the center.

Article Snippet: For chronic supplementation with IFNλ2 and/or IFNλ3, IFNλ2/3 KO cells were seeded in the presence of 5 ng/mL IFNλ2 (R&D Systems #1587IL025/CF) and 1 ng/mL IFNλ3 (R&D Systems #5259-IL-025/CF).

Techniques: Recombinant, Infection, Luciferase, Immunofluorescence, Staining, Live Cell Imaging, Control, Standard Deviation

Journal: PLOS Pathogens

Article Title: Basal IFNλ2/3 signaling is required for ISG expression and viral control in human intestinal epithelial cells

doi: 10.1371/journal.ppat.1013857

Figure Lengend Snippet: T84 WT, IFNλ1 KO, and IFNλ2/3 KO cells were seeded in 48-well plates and infected the following day. (A) Cells were infected with VSV-GFP (MOI = 1), and infection was assessed at 7 hours post-infection (hpi) by live-cell microscopy. Nuclei were stained with Hoechst (blue), and infected cells are shown in green. (B) Cells were infected with MRV (MOI = 1), and infection was evaluated at 16 hpi by immunostaining against the MRV μNS protein; nuclei were counterstained with DAPI. (C) Cells were infected with RV-UnaG (MOI = 1), and infection was measured by live-cell microscopy at 12 hpi. (D) Cells were infected with VV-GFP (MOI = 1), and infection was evaluated at 16 hpi using live-cell microscopy. (A–D) Representative images (left) and corresponding quantification (right) are shown for each virus. Scale bar = 100 μm. Data represent ≥3 independent biological replicates. Statistical significance was determined by two-way ANOVA (*P < 0.05, ****P < 0.0001, ns = not significant). Error bars represent standard deviation with the mean as the center.

Article Snippet: For chronic supplementation with IFNλ2 and/or IFNλ3, IFNλ2/3 KO cells were seeded in the presence of 5 ng/mL IFNλ2 (R&D Systems #1587IL025/CF) and 1 ng/mL IFNλ3 (R&D Systems #5259-IL-025/CF).

Techniques: Infection, Microscopy, Staining, Immunostaining, Virus, Standard Deviation

Journal: PLOS Pathogens

Article Title: Basal IFNλ2/3 signaling is required for ISG expression and viral control in human intestinal epithelial cells

doi: 10.1371/journal.ppat.1013857

Figure Lengend Snippet: (A–H) T84 WT, IFNLR KO, IFNλ1 KO, and IFNλ2/3 KO cells were seeded in (A, B, E, G) 48-well plate as 200,000 cell/well or (C, D, F, H) 98-well plate as 50,000 cell/well, and next day the media was replaced with 20 μM H151 (STING inhibitor) or DMSO (solvent control). Cells were incubated with H151 or DMSO for 2 days and subsequently infected with VSV-Luc (MOI = 1) for 7 hours in the continued presence or absence of H151. (A, B, E, G) Basal and virus-induced IFNλ1 and/or IFNλ2/3 expression was assessed by qRT-PCR. (C, D, F, H) Virus infection was quantified by luciferase assay. Relative expression was normalized to TBP. Data represent n ≥ 3 biological replicates. Statistical significance was determined using one-way ANOVA with multiple comparisons (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns = not significant). Error bars represent standard deviation with the mean shown at the center.

Article Snippet: For chronic supplementation with IFNλ2 and/or IFNλ3, IFNλ2/3 KO cells were seeded in the presence of 5 ng/mL IFNλ2 (R&D Systems #1587IL025/CF) and 1 ng/mL IFNλ3 (R&D Systems #5259-IL-025/CF).

Techniques: Solvent, Control, Incubation, Infection, Virus, Expressing, Quantitative RT-PCR, Luciferase, Standard Deviation

Journal: PLOS Pathogens

Article Title: Basal IFNλ2/3 signaling is required for ISG expression and viral control in human intestinal epithelial cells

doi: 10.1371/journal.ppat.1013857

Figure Lengend Snippet: T84 WT, IFNλ1 KO, IFNλ2/3 KO, and IFNLR KO cells were seeded in 48-well plates and subjected to RNA sequencing three days post-seeding. (A) Principal Component Analysis (PCA) plot displaying the distribution of T84 WT, IFNλ1 KO, IFNλ2/3 KO, and IFNLR KO cells based on their gene expression profiles. Each point represents an individual sample, colored according to the experimental group. (B) T84 IFNLR KO vs. WT cells, (C) T84 IFNλ1 KO vs. WT cells, (D) T84 IFNλ2/3 KO vs. WT cells. (B-D) Each point represents a gene, plotted by its fold-change (x-axis) and statistical significance (-log10 p-value, y-axis). Genes with significant differential expression ( p < 0.05) are highlighted in black (upregulated) and green, blue and red (downregulated). The most downregulated genes in KO cells are labeled. (E) Gene Ontology (GO) enrichment analysis was performed for Biological Process (BP) terms using the top 500 differentially expressed genes (DEGs) from each WT vs. KO cells comparison. The heatmap displays the top 30 GO terms ranked by their average significance score, and hierarchically clustered based on the similarity of their enrichment profiles. The color intensity represents the statistical significance of each GO term’s enrichment, calculated as the − log 10 (p-value). (F) The heatmap displays the top 25 differentially expressed genes associated with the biological process “innate immune response” (GO:0045087). Rows represent genes, columns represent samples, and hierarchical clustering was applied to both. Color intensity indicates relative expression levels (red: high; blue: low). Asterisk-marked genes are further validated in and . Data represents three independent biological replicates.

Article Snippet: For chronic supplementation with IFNλ2 and/or IFNλ3, IFNλ2/3 KO cells were seeded in the presence of 5 ng/mL IFNλ2 (R&D Systems #1587IL025/CF) and 1 ng/mL IFNλ3 (R&D Systems #5259-IL-025/CF).

Techniques: RNA Sequencing, Gene Expression, Quantitative Proteomics, Labeling, Comparison, Expressing

Journal: PLOS Pathogens

Article Title: Basal IFNλ2/3 signaling is required for ISG expression and viral control in human intestinal epithelial cells

doi: 10.1371/journal.ppat.1013857

Figure Lengend Snippet: (A) qRT-PCR analysis of select ISGs Mx1, OAS1, ISG15, IRF7, RIG-I, and IFIT1 in T84 WT, IFNλ1 KO, IFNλ2/3 KO, and IFNLR KO three days post-seeding. Relative expression was normalized to TBP. (B) Western blot analysis of select ISGs (Mx1, IRF7, RIG-I, ISG15 and STAT1) in T84 WT, IFNλ1 KO, IFNλ2/3 KO, and IFNLR KO three days post-seeding. Mx1, IRF7, RIG-I, ISG15 and STAT1 protein abundance was quantified relative to actin as loading control. Representative images shown. (C) T84 WT, IFNλ1 KO, IFNλ2/3 KO cells were treated with recombinant IFNl1-3 proteins (100ng/mL) and cells were collected at 0-, 1-, 3-, and 6-hours post-treatment. Western Blot analysis of p-STAT1 and STAT1 was performed. P-STAT1 and STAT1 abundances were quantified relative to actin as loading control. Representative images shown. (D) Same as (C) but ISG (Mx1, OAS1, ISG15 and IFIT1) induction was assessed by qRT-PCR 24 h post-treatment. Relative expression was normalized to TBP. Data represent n ≥ 3 biological replicates. Statistical significance was determined using two-way ANOVA (*P < 0.05, P < 0.01 **, P < 0.001 ***, P < 0.0001 ****, ns = not significant). Error bars represent standard deviation with the mean as the center.

Article Snippet: For chronic supplementation with IFNλ2 and/or IFNλ3, IFNλ2/3 KO cells were seeded in the presence of 5 ng/mL IFNλ2 (R&D Systems #1587IL025/CF) and 1 ng/mL IFNλ3 (R&D Systems #5259-IL-025/CF).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Quantitative Proteomics, Control, Recombinant, Standard Deviation

Journal: PLOS Pathogens

Article Title: Basal IFNλ2/3 signaling is required for ISG expression and viral control in human intestinal epithelial cells

doi: 10.1371/journal.ppat.1013857

Figure Lengend Snippet: (A–C) T84 WT, IFNλ1 KO, IFNλ2/3 KO, and IRF3 KO cells were seeded in 6 well plates as 2x10 6 cells/well, and the media was changed the following day with 1.5 mL fresh media. Two days later, the cell supernatant was collected after centrifugation at 2000rpm for 5 minutes (referred as conditioned media), and used to treat T84 WT and IFNLR KO cells. Cells were treated with culture media (DMEM-F12) as control. (A) Schematic representation of experimental design was created in BioRender Keser,Y. (2025) https://BioRender.com/6ln3qq4 . (B) At 1-hour post-treatment (hpt), cells were harvested for Western blot analysis of STAT1 phosphorylation. P-STAT1 protein abundance was quantified relative to total actin, loading control. Representative images shown.(C) At 24 hours post-treatment, cells were harvested to assess ISG induction. qRT-PCR analysis of ISGs (Mx1, IFIT1, and ISG15) was performed following treatment by conditioned media. Relative expression was normalized to TBP. Data represent n ≥ 3 biological replicates. Statistical significance was determined using two-way ANOVA (*P < 0.05, P < 0.01 **, P < 0.001 ***, P < 0.0001 ****, ns = not significant). Error bars represent standard deviation with the mean as the center.

Article Snippet: For chronic supplementation with IFNλ2 and/or IFNλ3, IFNλ2/3 KO cells were seeded in the presence of 5 ng/mL IFNλ2 (R&D Systems #1587IL025/CF) and 1 ng/mL IFNλ3 (R&D Systems #5259-IL-025/CF).

Techniques: Centrifugation, Control, Western Blot, Phospho-proteomics, Quantitative Proteomics, Quantitative RT-PCR, Expressing, Standard Deviation

Journal: PLOS Pathogens

Article Title: Basal IFNλ2/3 signaling is required for ISG expression and viral control in human intestinal epithelial cells

doi: 10.1371/journal.ppat.1013857

Figure Lengend Snippet: (A–F) T84 WT, IFNλ1 KO, IFNλ2/3 KO, and IRF3 KO cells were seeded in 6 well plates as 2x10 6 cells/well, and the media was replaced the following day with 1.5 mL fresh media. Two days later, the cell supernatant was collected after centrifugation at 2000rpm for 5 minutes (referred to as conditioned media), and used to treat T84 IRF3 KO cells for 24 hours. Cells treated with culture media (DMEM-F12) served as a control. At 24 h post-treatment, cells were infected. (A) Schematic representation of experimental design was created in BioRender Keser,Y. (2025) https://BioRender.com/f9bbe51 . (B, C) VSV-GFP, (D) VSV_Luc, and (E, F) RV-UnaG. (B) VSV-GFP infection was assessed by live-cell imaging at 7 hpi, with nuclei stained using Hoechst. (C) Quantification of B. (C) VSV-Luc replication was assessed by luciferase assay at 7 hpi. (D) RV-UnaG infection (16 hpi) was evaluated by live-cell imaging, with nuclei stained using Hoechst. (F) Quantification of E. (B, E) Representative images shown. Scale bar = 100 μm. Data represent n ≥ 3 biological replicates. Statistical significance was determined using two-way ANOVA ( P < 0.05 *, P < 0.01 **, P < 0.001 ***, P < 0.0001 ****, ns = not significant). Error bars represent standard deviation with the mean as the center.

Article Snippet: For chronic supplementation with IFNλ2 and/or IFNλ3, IFNλ2/3 KO cells were seeded in the presence of 5 ng/mL IFNλ2 (R&D Systems #1587IL025/CF) and 1 ng/mL IFNλ3 (R&D Systems #5259-IL-025/CF).

Techniques: Centrifugation, Control, Infection, Live Cell Imaging, Staining, Luciferase, Standard Deviation

Journal: PLOS Pathogens

Article Title: Basal IFNλ2/3 signaling is required for ISG expression and viral control in human intestinal epithelial cells

doi: 10.1371/journal.ppat.1013857

Figure Lengend Snippet: (A) Schematic of the conditioned-media (CM) neutralization workflow was created in BioRender Keser,Y. (2025) https://BioRender.com/drh0ch2 . T84 WT cells were seeded in 6 well plates as 2x10 6 cells/well, and the media was replaced the following day with 1.5 mL fresh media. Two days later, the cell supernatant was collected after centrifugation at 2000rpm for 5 minutes (referred to as conditioned media (CM)). This conditioned media were incubated with neutralizing antibodies targeting IFNλ1 (α-λ1), IFNλ2 (α-λ2), IFNλ3 (α-λ3), IFNλ2/3 (α-λ2/3), or all three subtypes (α-λ1/2/3) for 1 h at room temperature. Antibody-treated CM were applied to T84 WT cells for analysis of STAT1 phosphorylation (1 h post-treatment) and ISG expression (16 h post-treatment). (B) Representative Western blots showing pSTAT1, total STAT1, and actin as a loading control following treatment with antibody-depleted CM. p-STAT1 protein abundance was quantified relative to STAT1. (C) qRT-PCR analysis of MX1 expression (normalized to TBP) 16 h after antibody-depleted CM treatment. (D) Same as A except CM were used to pre-treat T84 IRF3-KO cells for 24 h prior to VSV-Luc (MOI = 1) infection to assess antiviral activity at 7 hpi. Created in BioRender Keser,Y. (2025) https://BioRender.com/1zuiu9o . (E) VSV-Luciferase assay in T84 IRF3-KO cells pre-treated with antibody-depleted CM at 7 hpi. Data represent n ≥ 3 biological replicates. Statistical significance was determined using one-way ANOVA with multiple-comparison correction (*P < 0.05, P < 0.01 **, P < 0.001 ***, P < 0.0001 ****, ns = not significant). Error bars represent standard deviation with the mean as the center.

Article Snippet: For chronic supplementation with IFNλ2 and/or IFNλ3, IFNλ2/3 KO cells were seeded in the presence of 5 ng/mL IFNλ2 (R&D Systems #1587IL025/CF) and 1 ng/mL IFNλ3 (R&D Systems #5259-IL-025/CF).

Techniques: Neutralization, Centrifugation, Incubation, Phospho-proteomics, Expressing, Western Blot, Control, Quantitative Proteomics, Quantitative RT-PCR, Infection, Activity Assay, Luciferase, Comparison, Standard Deviation

Journal: PLOS Pathogens

Article Title: Basal IFNλ2/3 signaling is required for ISG expression and viral control in human intestinal epithelial cells

doi: 10.1371/journal.ppat.1013857

Figure Lengend Snippet: (A, B) T84 WT cells were seeded, and media was replaced the following day. After 48 h, supernatants (conditioned media) were collected and used as a reference control for antiviral activity. IRF3 KO cells were treated with recombinant IFNλ2 or IFNλ3 (0.01–20 ng/mL) or with WT conditioned media for 24 h and then infected with VSV-Luc for 7 h. (A) Schematic representation of the experimental workflow was created in BioRender Keser,Y. (2025) https://BioRender.com/ip2l074 . (B) 7hpi luciferase activity was measured to assess VSV-Luc infection in IRF3 KO cells treated with recombinant IFNλ2 or IFNλ3. (C–F) IFNλ2/3 KO cells were chronically supplemented for two weeks with IFNλ2 (5 ng/mL), IFNλ3 (1 ng/mL), or both. Cells were then trypsinized, reseeded in the absence of any IFN treatment and collected 48 h later for ISG analysis, or used for antiviral assays. (C) Schematic representation of chronic IFNλ2/3 supplementation and subsequent experimental steps. Created in BioRender Keser,Y. (2025) https://BioRender.com/3775duy . (D) Western blot analysis of IRF7, RIG-I, and STAT1 in WT cells and IFNλ2/3 KO cells under the indicated supplementation conditions or non-treated (NT). Protein abundance was quantified relative to actin. Representative images are shown. (E) qRT-PCR analysis of ISGs (MX1, IFIT1, OAS1) in WT cells and IFNλ2/3 cells maintained with IFNλ2, IFNλ3, IFNλ2 + 3, or non-treated. Relative expression was normalized to TBP. (F) VSV-Luc infection was measured by luciferase assayed 7 hpi in hours in WT and IFNλ2/3 cells maintained with IFNλ2, IFNλ3, IFNλ2 + 3, or non-treated. (G–I) IFNλ2/3 KO cells were chronically supplemented with IFNλ2 (5 ng/mL), IFNλ3 (1 ng/mL), or IFNλ2 + 3 for two weeks, reseeded in the absence of any IFNs, and next day, acutely stimulated with IFNλ1–3 (20 ng/mL of each) for 1 h or 24 h. (G) Schematic representation of chronic supplementation followed by acute IFNλ stimulation, was created in BioRender Keser,Y. (2025) https://BioRender.com/beodbxz . (H) Western blot analysis of p-STAT1 and total STAT1 in WT and IFNλ2/3 cells maintained with IFNλ2, IFNλ3, IFNλ2 + 3, or non-treated (NT). Protein abundance was quantified relative to actin, loading control. Representative images are shown. (I) qRT-PCR analysis of ISGs (MX1, IFIT1, OAS1) 24 h after acute IFNλ1–3 stimulation in WT and ΔIFNλ2/3 cells supplemented as indicated. Relative expression was normalized to TBP. Data represent n ≥ 3 biological replicates. Statistical significance was determined using two-way ANOVA (P < 0.05 *, P < 0.01 **, P < 0.001 ***, P < 0.0001 ****, ns = not significant). Error bars represent standard deviation, with the mean shown at the center.

Article Snippet: For chronic supplementation with IFNλ2 and/or IFNλ3, IFNλ2/3 KO cells were seeded in the presence of 5 ng/mL IFNλ2 (R&D Systems #1587IL025/CF) and 1 ng/mL IFNλ3 (R&D Systems #5259-IL-025/CF).

Techniques: Control, Activity Assay, Recombinant, Infection, Luciferase, Western Blot, Quantitative Proteomics, Quantitative RT-PCR, Expressing, Standard Deviation

Journal: PLOS Pathogens

Article Title: Basal IFNλ2/3 signaling is required for ISG expression and viral control in human intestinal epithelial cells

doi: 10.1371/journal.ppat.1013857

Figure Lengend Snippet: In WT cells (left panel), both IFNλ1 and IFNλ2/3 are produced under homeostatic conditions via IRF3 activation. Secreted IFNλs engage the IFNLR receptor on neighboring cells, activating the JAK/STAT pathway and inducing robust expression of ISGs thereby limiting viral replication. In IFNλ1 KO cells (middle panel), IFNλ2/3 are still expressed and can activate STAT1/2 signaling and ISG expression, maintaining effective antiviral defense with only a minor reduction in ISG levels. In contrast, IFNλ2/3 KO cells (right panel) retain IFNλ1 expression but exhibit a dramatic loss of STAT1/2 expression and fail to activate ISG transcription, resulting in impaired JAK/STAT signaling and increased viral replication. These findings highlight the predominant and non-redundant role of IFNλ2/3 in establishing and sustaining the basal antiviral state in intestinal epithelial cells. Schematics were created in BioRender Keser,Y. (2025) https://BioRender.com/3oi4mf0 .

Article Snippet: For chronic supplementation with IFNλ2 and/or IFNλ3, IFNλ2/3 KO cells were seeded in the presence of 5 ng/mL IFNλ2 (R&D Systems #1587IL025/CF) and 1 ng/mL IFNλ3 (R&D Systems #5259-IL-025/CF).

Techniques: Produced, Activation Assay, Expressing