t84 knock out ko cells  (ATCC)

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: Wild type (WT) T84 (ATCC CCL-248) as well as T84 knock-out (KO) cells were cultured in a 50:50 mixture of Dulbecco’s Modified Eagle’s Medium (DMEM) and F12 (Gibco #11320033).

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: Wild type (WT) T84 (ATCC CCL-248) as well as T84 knock-out (KO) cells were cultured in a 50:50 mixture of Dulbecco’s Modified Eagle’s Medium (DMEM) and F12 (Gibco #11320033).

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: Wild type (WT) T84 (ATCC CCL-248) as well as T84 knock-out (KO) cells were cultured in a 50:50 mixture of Dulbecco’s Modified Eagle’s Medium (DMEM) and F12 (Gibco #11320033).

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: Wild type (WT) T84 (ATCC CCL-248) as well as T84 knock-out (KO) cells were cultured in a 50:50 mixture of Dulbecco’s Modified Eagle’s Medium (DMEM) and F12 (Gibco #11320033).

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: Wild type (WT) T84 (ATCC CCL-248) as well as T84 knock-out (KO) cells were cultured in a 50:50 mixture of Dulbecco’s Modified Eagle’s Medium (DMEM) and F12 (Gibco #11320033).

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: Wild type (WT) T84 (ATCC CCL-248) as well as T84 knock-out (KO) cells were cultured in a 50:50 mixture of Dulbecco’s Modified Eagle’s Medium (DMEM) and F12 (Gibco #11320033).

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: Wild type (WT) T84 (ATCC CCL-248) as well as T84 knock-out (KO) cells were cultured in a 50:50 mixture of Dulbecco’s Modified Eagle’s Medium (DMEM) and F12 (Gibco #11320033).

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: Wild type (WT) T84 (ATCC CCL-248) as well as T84 knock-out (KO) cells were cultured in a 50:50 mixture of Dulbecco’s Modified Eagle’s Medium (DMEM) and F12 (Gibco #11320033).

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: Wild type (WT) T84 (ATCC CCL-248) as well as T84 knock-out (KO) cells were cultured in a 50:50 mixture of Dulbecco’s Modified Eagle’s Medium (DMEM) and F12 (Gibco #11320033).

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: Wild type (WT) T84 (ATCC CCL-248) as well as T84 knock-out (KO) cells were cultured in a 50:50 mixture of Dulbecco’s Modified Eagle’s Medium (DMEM) and F12 (Gibco #11320033).

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