recombinant human ifnλ1  (PeproTech)

Journal: Nature Immunology

Article Title: Bat organoids reveal antiviral responses at epithelial surfaces

doi: 10.1038/s41590-025-02155-1

Figure Lengend Snippet: a , Venn diagrams (generated using eulerr.co ) showing the number of significantly upregulated genes determined by bulk RNA-seq in bat nasal ORG , alv ORG , or SI ORG (n = 3 each) following treatment with universal IFNα2 (uIFNα2) or IFNλ1-like compared to mock-treated. The total number of upregulated genes per treatment are indicated. Overlapping regions represent genes commonly induced by both IFNs. b , RT-qPCR analysis of IFIT3 and ISG15 expression, normalized to EEF1A1 (2 –ΔCT ), in bat nasal ORG (n = 3) treated with uIFNα2 or IFNλ1-like for 8 h, with or without ruxolitinib (n = 3). Two-sided unpaired Student’s t-tests were used to compare mRNA levels between ruxolitinib-treated and mock-treated, interferon-stimulated nasal ORG . (ns: not significant; *** P < 0.001, **** P < 0.0001). c , Gene ontology (GO) enrichment analysis of bulk RNA-seq data, performed with clusterProfiler , revealed biological processes significantly enriched among genes upregulated in uIFNα2 vs. mock-infected bat nasal ORG , alv ORG , or SI ORG after 8 h (n = 3 each). The top 10 enriched biological processes are shown. The x axis indicates the ratio of enriched to total genes within each GO term. Dot size reflects the number of enriched genes per pathway. d , Same as in (c) but for IFNλ1-like vs. mock-treated bat nasal ORG , alv ORG , or SI ORG . e , Scatter plot showing log 2 -fold changes in gene expression from bulk RNA-seq comparing human SI ORG treated with uIFNα2 ( x axis) or IFNλ1-like ( y axis) to mock-treated controls after 8 h (n = 3). Individual ISGs and selected pro-inflammatory cytokines (red) are highlighted and labeled. f , same as in (c) but for uIFNα2 (left) or IFNλ1 (right) vs. mock-treated human SI ORG . g , Same as in (e) but for human bronchial ALI (n = 3). h , Same as in (f) but for GO enrichment analysis of uIFNα2 (left) or IFNλ1 (right) vs. mock-treated human bronchial ALI .

Article Snippet: After 1–2 days after seeding, diluted amounts of recombinant bat IFNλ (dose equivalent to 1,000 U ml −1 of universal IFNα2), 1,000 U ml −1 of universal IFNα2 or 100 ng ml −1 of recombinant human IFNλ1 (PeproTech) were added for 8 h unless otherwise indicated.

Techniques: Generated, RNA Sequencing, Quantitative RT-PCR, Expressing, Infection, Gene Expression, Labeling

Journal: Nature Immunology

Article Title: Bat organoids reveal antiviral responses at epithelial surfaces

doi: 10.1038/s41590-025-02155-1

Figure Lengend Snippet: a , Heatmap showing gene expression log 2 -fold changes (from EdgeR bulk RNA-seq differential gene expression analysis) of selected pro-inflammatory genes and selected ISGs in universal IFNα2 (uIFNα2) or IFNλ1 vs. mock-treated human SI ORG or human Bronchial ALI (n = 3 each). b , Same as for (a) but for IFN-treated bat SI ORG (n = 3), bat alv ORG (n = 3), or bat nasal ORG (n = 3). c , Same as for (a) but for MARV-, MERS-CoV- or SeV-infected bat alv ORG (n = 3 each), SeV-infected bat SI ORG (n = 3), or MARV-infected bat nasal ALI (n = 3). Comparisons used for calculation of edgeR log 2 -fold change are indicated in the legend.

Article Snippet: After 1–2 days after seeding, diluted amounts of recombinant bat IFNλ (dose equivalent to 1,000 U ml −1 of universal IFNα2), 1,000 U ml −1 of universal IFNα2 or 100 ng ml −1 of recombinant human IFNλ1 (PeproTech) were added for 8 h unless otherwise indicated.

Techniques: Gene Expression, RNA Sequencing, Infection

Journal: Nature Immunology

Article Title: Bat organoids reveal antiviral responses at epithelial surfaces

doi: 10.1038/s41590-025-02155-1

Figure Lengend Snippet: a , Scatter plots showing the log 2 -transformed fold changes in gene expression from bulk RNA-seq comparing nasal ORG (left), alv ORG (middle) or SI ORG (right) treated with universal IFNα2 ( x axis) or IFNλ1-like ( y axis) to mock-treated controls after 8 h. Individual ISGs are highlighted. IFNL3 -like ( LOC107521776 ) is highlighted in red. b , RT–qPCR analysis of IFNL3 -like ( LOC107521776 ) in bat SI ORG or alv ORG ; IFNL3 in human SI ORG ) normalized to EEF1A1 (2 −Δ C t ) after treatment with bat IFNλ1-like (bat organoids) or human IFNλ1 (human SI ORG ) for 4 and 8 h. A two-sided Student’s t -test was used to compare IFNL3 -like mRNA levels at 4 or 8 h after treatment to mock-treated controls. c , Scatter plot showing the log 2 -transformed fold changes in gene expression from bulk RNA-seq comparing SI ORG treated with universal IFNα2 ( x axis) or IFNλ3-like (derived from LOC107521776 ) ( y axis) to mock-treated controls after 8 h. Individual ISGs are highlighted in red. d , RT–qPCR analysis of IFNL3 -like ( LOC107521776 ) or MX1 (normalized to EEF1A1 , 2 − Δ C t ) in bat SI ORG engineered with Cas9 and IRF9 targeting (sgIRF9) or control (sgScrambled) RNA, treated with or without bat IFNλ1-like for 8 h. A two-sided Student’s t -test was used to compare IFNL3 -like mRNA levels between sgIRF9 and sgScrambled SI ORG at 8 h after treatment. e , As in d but comparing sgIRF9 and sgScrambled SI ORG after infection with VSV-eGFP at an MOI of 0.05 for 72 h. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: After 1–2 days after seeding, diluted amounts of recombinant bat IFNλ (dose equivalent to 1,000 U ml −1 of universal IFNα2), 1,000 U ml −1 of universal IFNα2 or 100 ng ml −1 of recombinant human IFNλ1 (PeproTech) were added for 8 h unless otherwise indicated.

Techniques: Transformation Assay, Gene Expression, RNA Sequencing, Quantitative RT-PCR, Derivative Assay, Control, Infection

Journal: Nature Immunology

Article Title: Bat organoids reveal antiviral responses at epithelial surfaces

doi: 10.1038/s41590-025-02155-1

Figure Lengend Snippet: a , Experimental workflow showing the administration of universal IFNα2, IFNλ1-like or IFNλ3-like 12 h before, during or 8 h after treatment with VSV infection of bat nasal ORG or SI ORG . Intracellular VSV viral RNA was measured 24 h after infection. b , c , RT–qPCR analysis of intracellular VSV-NP viral RNA in bat SI ORG ( n = 3) ( b ) or nasal ORG ( n = 3) ( c ), normalized to EEF1A1 (2 −Δ C t ) and expressed as a percentage relative to no IFN control. A two-sided Student’s t -test was used to compare VSV viral RNA levels across before, during and after treatment, and no IFN conditions, comparing each IFN treatment to no IFN control. d , Experimental workflow showing bat nasal ORG or SI ORG treated with universal IFNα2 or bat IFNλ1-like for 3 h, followed by washout and incubation in IFN-free medium for 24 h and RNA collection at 0, 3 and 24 h after IFN washout. e , f , RT–qPCR analysis of ISGs (normalized to EEF1A1 ) in bat SI ORG ( n = 3) ( e ) or nasal ORG ( n = 3) ( f ) treated with universal IFNα2 or bat IFNλ1-like at 3 h (left) or 24 h (right) after IFN removal. A two-sided Student’s t -test was used to compare ISG mRNA levels between treatments. g , RT–qPCR analysis of intracellular SINV-eGFP nsP2-P726G nsP1 RNA (normalized to EEF1A1 (2 −Δ C t ) and expressed as fold change relative to the mean of the sgScrambled control) in bat SI ORG engineered with Cas9 and targeted guide RNA (sgIRF9, sgIFNAR2, sgIFNLR1) or control (sgScrambled), infected with SINV-eGFP nsP2-P726G . A two-sided Student’s t -test was used to compare SINV nsP 1 viral RNA levels between treatment groups. nsP1, nonstructural protein 1. h , RT–qPCR analysis of intracellular MARV-L viral RNA (normalized to EEF1A1) in bat SI ORG , nasal ORG or alv ORG ( n = 4) treated with or without IFNs during a 3-day MARV infection. A two-sided Student’s t -test was used to compare MARV viral RNA levels between IFN-treated and untreated controls. i , RT–qPCR analysis of MARV-L viral RNA in bat alv ORG treated with or without 5 µM ruxolitinib during a 3-day infection. A two-sided Student’s t -test was used to compare MARV viral RNA levels between ruxolitinib and dimethylsulfoxide (DMSO) mock-treated alv ORG and MARV -infected alv ORG . * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: After 1–2 days after seeding, diluted amounts of recombinant bat IFNλ (dose equivalent to 1,000 U ml −1 of universal IFNα2), 1,000 U ml −1 of universal IFNα2 or 100 ng ml −1 of recombinant human IFNλ1 (PeproTech) were added for 8 h unless otherwise indicated.

Techniques: Infection, Quantitative RT-PCR, Control, Incubation

Journal: Nature Immunology

Article Title: Bat organoids reveal antiviral responses at epithelial surfaces

doi: 10.1038/s41590-025-02155-1

Figure Lengend Snippet: a , Experimental workflow to assess the temporal induction and maintenance of ISG expression. Bat Nasal ORG or SI ORG are treated with universal IFNα2 (uIFNα2) or bat IFNλ1-like for 3 hours, followed by washout and incubation in IFN-free medium for 24 hours. b , Heatmaps showing RT-qPCR analysis of selected ISGs in bat SI ORG (n = 3), with expression values calculated using the 2 - ΔΔCT method: first normalized to EEF1A1 (2 –ΔCT ), then to baseline levels at −3 h (before treatment). Timepoints shown include before IFN treatment (−3 h), immediately after treatment (0 h), and various hours post-interferon removal (+6h, +24h). Each heatmap value represents the average normalized ISG expression across three biological replicates. Left, uIFNα2. Right, IFNλ1-like treatment. c , same as in (b) but for bat nasal ORG . d , RT-qPCR analysis of IFIT1 (normalized to EEF1A1 (2 –ΔCT ), then expressed as a percentage relative to the average sgScrambled control in bat nasal ORG engineered with Cas9 and a targeted guide RNA (sgIRF9, sgIFNAR2 or sgIFNLR1) or control guide RNA (sgScrambled) (n = 3 each), treated or not for 8 h with uIFNα2 or bat IFNλ1-like.

Article Snippet: After 1–2 days after seeding, diluted amounts of recombinant bat IFNλ (dose equivalent to 1,000 U ml −1 of universal IFNα2), 1,000 U ml −1 of universal IFNα2 or 100 ng ml −1 of recombinant human IFNλ1 (PeproTech) were added for 8 h unless otherwise indicated.

Techniques: Expressing, Incubation, Quantitative RT-PCR, Control

Journal: Molecular Systems Biology

Article Title: The population context is a driver of the heterogeneous response of epithelial cells to interferons

doi: 10.1038/s44320-024-00011-2

Figure Lengend Snippet: ( A ) Schematic depicting the T84 prom-Mx1-fp reporter cell line. Upon interaction of IFNs with their receptor, downstream signaling induces nuclear translocation of the transcription complex ISGF3. This leads to expression of the fluorescent protein under control of the ISG Mx1 promoter. The fluorescent protein accumulates in the cytosol and can be visualized by fluorescence microscopy. ( B ) Representative images showing expression of the fluorescent reporter (white) after mock, 2000 IU/mL IFNβ1, or 300 ng/mL IFNλ1-3 treatment. Nuclei are stained with DAPI (blue). n = 3 biological replicates. Scale bar = 100 µm. ( C ) T84-prom-Mx1-fp seeded at medium density were mock treated or treated with 2000 IU/mL IFNβ1 or 300 ng/mL IFNλ1-3 for 24 h. The positive fluorescent cells were determined for each edge degree and the mean fluorescence intensity (MFI) was measured within each positive cell. The MFI was normalized to the mock-treatment MFI of the corresponding edge degree (normalized fluorescence). ( D ) T84-prom-Mx1-fp seeded at medium density were mock treated or treated with 2000 IU/mL IFNβ1 or 300 ng/mL IFNλ1-3 for 6, 12, 24, 48, 72, and 96 h. Quantification of the percentage of total positive fluorescent cells as compared to mock-treated cells. ( E , F ) Ileum-derived organoids were seeded in 2-dimensions (2D) and treated apically with 2000 IU/mL IFNβ1 or 300 ng/mL IFNλ1-3. 24 h post-treatment, samples were fixed and indirect immunofluorescence was performed against ISG15 (green). Nuclei were stained with DAPI (blue). ( E ) Representative images are shown. Yellow arrows point at IFN responder cells located at the colony edges. Red arrows point at non-responder cells in the colony center. Scale bar = 100 µm. ( F ) Quantification of the ISG mean fluorescence intensity (arbitrary units (a.u.)) at the edge or the center of cell clusters. ( D , F ) n ≥ 3 biological replicates, error bars indicate the standard deviation. n.s. = not significant. P < 0.05 *, P < 0.01 **, P < 0.001 ***, P < 0.0001 **** as determined by ( D ) ordinary one-way ANOVA with Dunnett’s multiple comparison test using edge degree 1 as reference and ( F ) Unpaired t test with Welch’s correction.

Article Snippet: Human recombinant IFNλ1 (IL-29) (#300-02L), IFNλ2 (IL28A) (#300-2K), and IFNλ3 (IL-28B) (#300-2K) were purchased from Peprotech, and cells were treated by a cocktail of all three type III IFNs in a ratio of 1:1:1, resulting in a final concentration of 300 ng/mL or as described in the figure legend.

Techniques: Translocation Assay, Expressing, Control, Fluorescence, Microscopy, Staining, Derivative Assay, Immunofluorescence, Standard Deviation, Comparison

Journal: Molecular Systems Biology

Article Title: The population context is a driver of the heterogeneous response of epithelial cells to interferons

doi: 10.1038/s44320-024-00011-2

Figure Lengend Snippet: ( A – E ) T84-prom-Mx1-fp seeded at medium density were mock treated or treated with 2000 IU/mL IFNβ1 or 300 ng/mL IFNλ1-3 for 24 h. Cell nuclei were stained with DAPI and fluorescence microscopy was performed. ( A ) Representative images of cells (nuclei stained with DAPI are blue) expressing the fluorescent reporter (white). Yellow arrows point at IFN responder single cells or cells located at the colony edges. Red arrows point at non-responder cells in the colony center. ( B ) Correlation between single cell location and IFN-responsiveness was assessed using DBSCAN-CellX. Schematics depicting how the tool annotates cells according to their location at the edge or at the center of a cluster, or according to their edge degree are shown. ( C , D ) Quantification of the percentage of positive fluorescent cells as compared to mock-treated cells. ( C ) Edge vs. center cells. ( D ) Percentage of positive fluorescent cells dependent on the edge degree. An edge degree of 0 define single cells (no neighbors) and edge degree 1 are cells at the border of a colony. The higher the edge degree, the larger the distance from the edge. Each dot represents the percentage of positive cells dependent on the edge degree for a single well averaged over 12 individual fields of view per well. ( E ) Regression analysis and coefficient of correlation (ρ) calculated for ( D ) using a two-tailed nonparametric Spearman correlation. ( F ) T84-prom-Mx1-fp seeded at medium density were mock treated or treated with 2000 IU/mL IFNβ1 or 300 ng/mL IFNλ1-3 for 6, 12, 24, 48, 72, and 96 h. Quantification of the percentage of positive fluorescent cells as compared to mock-treated cells for single, edge and center cells. ( C , D , F ) Error bars indicate standard deviations. n ≥ 3 biological replicates. n.s. = not significant. P < 0.05 *, P < 0.01 **, P < 0.001 ***, P < 0.0001 **** as determined by ( C ) Unpaired t test with Welch’s correction, ( D ) ordinary one-way ANOVA with Dunnett’s multiple comparison test using edge degree 1 as reference and ( F ) ordinary one-way ANOVA within each time-point. .

Article Snippet: Human recombinant IFNλ1 (IL-29) (#300-02L), IFNλ2 (IL28A) (#300-2K), and IFNλ3 (IL-28B) (#300-2K) were purchased from Peprotech, and cells were treated by a cocktail of all three type III IFNs in a ratio of 1:1:1, resulting in a final concentration of 300 ng/mL or as described in the figure legend.

Techniques: Staining, Fluorescence, Microscopy, Expressing, Two Tailed Test, Comparison

Journal: Molecular Systems Biology

Article Title: The population context is a driver of the heterogeneous response of epithelial cells to interferons

doi: 10.1038/s44320-024-00011-2

Figure Lengend Snippet: ( A ) Schematic depicting the glass micropatterning approach using a Quartz-mask. ( B – D ) T84-prom-Mx1-fp cells seeded on circular micropatterns (200 µm diameter) were mock treated or treated with 2000 IU/mL IFNβ1 or 300 ng/mL IFNλ1-3. Fluorescent imaging was performed at 0 h, 12 h, 24 h, 48 h, 72 h, and 96 h post treatment. ( B ) Representative images at 0 h, 12 h, and 24 h post-treatment. The red line represents the edge of the patterns. Expression of the fluorescent reporter is depicted in white. Scale bar = 100 µm. ( C ) The radial distribution of immune response was determined for the 24 h post-treatment. Each population was segmented in rings (14 µm radius) and the mean fluorescence intensity (MFI) was measured within these rings. The MFI was normalized to the mock-treatment MFI of the corresponding ring. Each dot is one cell population (seeded on one micropattern). ( D ) The reporter expression for each single population was quantified by measuring the MFI at the edge and the center of a population at 12, 24, 48, 72, and 96 h post-treatment, and normalizing it to the mock-treatment of the respective time-point. Each dot is one cell population (seeded on one micropattern), lines connect edge and center of the same cell population. ( C , D ) n ≥ 3 biological replicates, in ( C ) error bars indicate the standard deviation. n.s. = not significant. P < 0.05 *, P < 0.01 **, P < 0.001 ***, P < 0.0001 **** as determined by ( C ) RM one-was ANOVA using the edge (0–14 µm) as reference and ( D ) Paired t test. .

Article Snippet: Human recombinant IFNλ1 (IL-29) (#300-02L), IFNλ2 (IL28A) (#300-2K), and IFNλ3 (IL-28B) (#300-2K) were purchased from Peprotech, and cells were treated by a cocktail of all three type III IFNs in a ratio of 1:1:1, resulting in a final concentration of 300 ng/mL or as described in the figure legend.

Techniques: Imaging, Expressing, Fluorescence, Standard Deviation

Journal: Molecular Systems Biology

Article Title: The population context is a driver of the heterogeneous response of epithelial cells to interferons

doi: 10.1038/s44320-024-00011-2

Figure Lengend Snippet: T84 cells seeded at high and low density were mock treated, or treated with 2000 IU/mL IFNβ1 or 300 ng/mL IFNλ1-3. ( A ) Schematic depicting the experimental setup. ( B ) At 0, 6, 12, 24, 48, 36, and 72 h post IFN treatment, RNA was harvested to evaluate the transcription of the representative ISGs IFIT1 and Mx1 using RT-q-PCR. ISG relative expression was normalized to the mock-treated cells of the respective time-point (fold change). ( C , D ) At 0, 0.5, 1, 1.5, and 2 h post treatment, cellular protein extracts were collected to assess the phospho-STAT1 (pSTAT1) abundance by Western Blot. ( D ) For the 1 h post-treatment samples, pSTAT1 was quantified relative to the housekeeping protein α-tubulin. ( B , D ) n = 3 biological replicates, error bars indicate the standard deviation. n.s. = not significant. P < 0.05 *, P < 0.01 **, P < 0.001 ***, P < 0.0001 **** as determined by Unpaired t test with Welch’s correction. .

Article Snippet: Human recombinant IFNλ1 (IL-29) (#300-02L), IFNλ2 (IL28A) (#300-2K), and IFNλ3 (IL-28B) (#300-2K) were purchased from Peprotech, and cells were treated by a cocktail of all three type III IFNs in a ratio of 1:1:1, resulting in a final concentration of 300 ng/mL or as described in the figure legend.

Techniques: Expressing, Western Blot, Standard Deviation

Journal: Molecular Systems Biology

Article Title: The population context is a driver of the heterogeneous response of epithelial cells to interferons

doi: 10.1038/s44320-024-00011-2

Figure Lengend Snippet: T84-prom-Mx1-fp cells at high or low density were treated with increasing concentrations of IFNβ1 and IFNλ1-3. Live cell fluorescence imaging was performed at an interval of 2 h for 24 h. ( A ) Representative images for selected time-points showing expression of the reporter prom-Mx1-mCherry (pMx1-mCh) in white. Nuclei are visualized by expression of H2B-turqiouse. Scale bar = 100 µm. ( B , C ) The mean fluorescence intensity (MFI) of the reporter expression within each cell was averaged for each density and normalized to the mock MFI of each time-point (fold change) for ( B ) IFNβ1 and ( C ) IFNλ1-3. n = 3 biological replicates. n.s = not significant, error bars indicate the standard deviation. P < 0.05 *, P < 0.01 **, P < 0.001 ***, P < 0.0001 **** as determined by Unpaired t test with Welch’s correction between high and low density for each time-point.

Article Snippet: Human recombinant IFNλ1 (IL-29) (#300-02L), IFNλ2 (IL28A) (#300-2K), and IFNλ3 (IL-28B) (#300-2K) were purchased from Peprotech, and cells were treated by a cocktail of all three type III IFNs in a ratio of 1:1:1, resulting in a final concentration of 300 ng/mL or as described in the figure legend.

Techniques: Fluorescence, Imaging, Expressing, Standard Deviation

Journal: Molecular Systems Biology

Article Title: The population context is a driver of the heterogeneous response of epithelial cells to interferons

doi: 10.1038/s44320-024-00011-2

Figure Lengend Snippet: ( A ) Epithelial and ( B ) non-epithelial cells were seeded at high (H) and low (L) density. Cells were mock treated, or treated with 2000 IU/mL IFNβ1 or 300 ng/mL IFNλ1-3. 24 h post IFN treatment, RNA was harvested to evaluate the transcription of the representative ISGs IFIT1 using RT-q-PCR. ISG relative expression was normalized to the mock-treated cells (fold change). n ≥ 3 biological replicates, error bars indicate the standard deviation. n.s. = not significant. P < 0.05 *, P < 0.01 **, P < 0.001 ***, P < 0.0001 **** as determined by Unpaired t test with Welch’s correction.

Article Snippet: Human recombinant IFNλ1 (IL-29) (#300-02L), IFNλ2 (IL28A) (#300-2K), and IFNλ3 (IL-28B) (#300-2K) were purchased from Peprotech, and cells were treated by a cocktail of all three type III IFNs in a ratio of 1:1:1, resulting in a final concentration of 300 ng/mL or as described in the figure legend.

Techniques: Expressing, Standard Deviation

Journal: Molecular Systems Biology

Article Title: The population context is a driver of the heterogeneous response of epithelial cells to interferons

doi: 10.1038/s44320-024-00011-2

Figure Lengend Snippet: ( A – D ) T84, Calu3, and human ileum-derived organoids seeded on transwell inserts were mock treated or treated from the apical (A) or basolateral (BL) side with 2000 IU/mL IFNβ1 or 300 ng/mL IFNλ1-3. ( A ) Schematic depicting the treatment of cells seeded on transwell inserts. ( B – D ) 24 h post treatment, RNA was harvested, and RT-q-PCR was used to evaluate the expression of the ISG IFIT1 and Mx1 for ( B ) T84 cells, ( C ) Calu3 cells, and ( D ) human ileum-derived organoids. Data is normalized to mock (fold change). ( E – G ) Organoids were grown in three-dimensionsional (3D) structures, either in a basolateral out (BL-out) or an apical out (A-out) conformation. ( E ) Schematic depicting BL-out and A-out organoids, and which membrane interacts with the IFNs during treatment. ( F ) BL-out and A-out organoids were stained for actin using Phalloidin (yellow), to mark the apical membrane of cells. Nuclei were stained with DAPI (blue). Scale bar = 100 µm. ( G ) BL-out and A-out organoids were treated with 2000 IU/mL IFNβ1 or 300 ng/mL IFNλ1-3. 24 h post treatment, RNA was harvested, and RT-q-PCR was used to evaluate the expression of the ISGs IFIT1 and Mx1. Data are normalized to mock (fold change). ( B – D , G ) n ≥ 3 biological replicates, error bars indicate the standard deviation. n.s. = not significant. P < 0.05 *, P < 0.01 **, P < 0.001 ***, P < 0.0001 **** as determined by Unpaired t test with Welch’s correction. .

Article Snippet: Human recombinant IFNλ1 (IL-29) (#300-02L), IFNλ2 (IL28A) (#300-2K), and IFNλ3 (IL-28B) (#300-2K) were purchased from Peprotech, and cells were treated by a cocktail of all three type III IFNs in a ratio of 1:1:1, resulting in a final concentration of 300 ng/mL or as described in the figure legend.

Techniques: Derivative Assay, Expressing, Membrane, Staining, Standard Deviation

Journal: Molecular Systems Biology

Article Title: The population context is a driver of the heterogeneous response of epithelial cells to interferons

doi: 10.1038/s44320-024-00011-2

Figure Lengend Snippet: ( A – C ) T84 cells were seeded on transwell inserts to allow for a polarized monolayer formation. ( A ) 5 days post seeding, cells were fixed, and indirect immunofluorescence was performed against the junctional complex protein ZO1 (green). Nuclei were stained with DAPI (blue). Representative image is shown. Scale bars = 50 µm. n = 3 biological replicates. ( B ) Formation and integrity of the monolayer was followed by measuring the transepithelial electrical resistance (TEER) (Ω/cm 2 ) over 5 days. Values > 1000 Ω/cm 2 (dotted line) shows that cells established a polarized monolayer formation. n = 3 biological replicates. ( C ) 5 days post seeding, after reaching a polarized monolayer, the integrity of the monolayer was confirmed by the FITC-Dextran permeability assay. Diffusion of FITC-Dextran from the apical to the basolateral compartment was measured and expressed as concentration (mg/mL) of FITC-Dextran in the basolateral compartment after 3 h incubation. Positive control (pos) was the maximum diffusion possible and the negative control (neg) was medium only without FITC-Dextran. ( D ) Micropatterning of transwell inserts: Schematic depicting the micropatterning on transwell membranes using the PRIMO system (Alvéole Lab, www.alveolelab.com ). ( E , F ) T84 prom-Mx1-fp cells were seeded on micropatterned transwell membranes. Cells were mock treated, or treated simultaneously from the apical and basolateral side with 2000 IU/mL IFNβ1 or 300 ng/mL IFNλ1-3. Cells were fixed at 0 h, 12 h, and 24 h post treatment and fluorescent imaging was performed. ( E ) Representatives images showing treated T84 cell populations. The red line represents the edge of the patterns. Expression of the fluorescent reporter is depicted in white. Scale bar = 100 µm. ( F ) The reporter expression was quantified by measuring the mean fluorescence intensity (MFI) at the edge and the center of a population at 12 h or 24 h post treatment, and normalizing it to the corresponding 0 h post treatment at the edge and center, respectively. Each dot is one cell population (seeded on one micropattern), lines connect edge and center of the same cell population. ( B , C , F ) n ≥ 3 biological replicates, in ( B , C ) error bars indicate the standard deviation. n.s. = not significant, P < 0.05 *, P < 0.01 **, P < 0.001 ***, P < 0.0001 **** as determined by ( B , C ) ordinary one-way ANOVA with Dunnett’s multiple comparison test using ( B ) day 1 or ( C ) the positive control as reference, and ( F ) Paired t test.

Article Snippet: Human recombinant IFNλ1 (IL-29) (#300-02L), IFNλ2 (IL28A) (#300-2K), and IFNλ3 (IL-28B) (#300-2K) were purchased from Peprotech, and cells were treated by a cocktail of all three type III IFNs in a ratio of 1:1:1, resulting in a final concentration of 300 ng/mL or as described in the figure legend.

Techniques: Immunofluorescence, Staining, FITC-Dextran Permeability Assay, Diffusion-based Assay, Concentration Assay, Incubation, Positive Control, Negative Control, Imaging, Expressing, Fluorescence, Standard Deviation, Comparison

Journal: Molecular Systems Biology

Article Title: The population context is a driver of the heterogeneous response of epithelial cells to interferons

doi: 10.1038/s44320-024-00011-2

Figure Lengend Snippet: ( A ) Schematic depicting paracellular diffusion in a monolayer of T84 WT and T84 ZO1 KO cells with disrupted junctional complexes. ( B ) T84 WT and T84 ZO1 KO cell protein extracts were harvested to control the absence of ZO1 protein in the KO cells by Western Blot. α-tubulin served as a housekeeping protein. Representative image is shown. ( C ) T84 WT and T84 ZO1 KO cells were fixed and indirect immunofluorescence was performed against the junctional complex protein ZO1 (green). Nuclei were stained with DAPI (blue). Representative image is shown. Scale bar = 100 µm. ( D ) T84 WT and T84 ZO1 KO cells were seeded on transwell inserts and grown as a polarized monolayer. Transepithelial electrical resistance (TEER) was measured over a period of 5 days. Dotted line shows a TEER of 1000 Ω/cm 2 corresponding to the resistance formed by confluent polarized T84 cells (Stanifer et al, ). ( E ) T84 WT and ZO1 KO cells were seeded on transwell inserts and grown as a dense monolayer, in which T84 WT cells reached a TEER > 1000 Ω/cm 2 . Cells were treated apically with 2000 IU/mL IFNβ1 and 300 ng/mL IFNλ1-3. 3 h after treatment, medium in the basolateral compartment was retrieved and IFN amount diffused from the apical to the basolateral transwell compartment was assessed by the HEK-blue assay. Depicted is the IFN concentration detected in the basolateral compartment for mock (m), IFNβ1 (β1) and IFNλ1-3 (λ) treated samples. ( F ) T84 WT and T84 ZO1 KO cells at high (H) and low (L) density were treated apically with 2000 IU/mL IFNβ1 or 300 ng/mL IFNλ1-3. 24 h post treatment, RNA was harvested, and RT-q-PCR was used to evaluate the expression of the ISG IFIT1. Data is normalized to mock (fold change). ( D – F ) n ≥ 3 biological replicates, error bars indicate the standard deviation. n.s. = not significant, P < 0.05 *, P < 0.01 **, P < 0.001 ***, P < 0.0001 **** as determined by ( D ) multiple t tests using the Bonferroni-Dunn method and analyzing WT and ZO1 KO conditions for each time-point individually, and ( E , F ) Unpaired t test with Welch’s correction. .

Article Snippet: Human recombinant IFNλ1 (IL-29) (#300-02L), IFNλ2 (IL28A) (#300-2K), and IFNλ3 (IL-28B) (#300-2K) were purchased from Peprotech, and cells were treated by a cocktail of all three type III IFNs in a ratio of 1:1:1, resulting in a final concentration of 300 ng/mL or as described in the figure legend.

Techniques: Diffusion-based Assay, Control, Western Blot, Immunofluorescence, Staining, Concentration Assay, Expressing, Standard Deviation

Journal: Molecular Systems Biology

Article Title: The population context is a driver of the heterogeneous response of epithelial cells to interferons

doi: 10.1038/s44320-024-00011-2

Figure Lengend Snippet: T84 cells seeded at high and low density were mock-treated or pre-treated with 2000 IU/mL IFNβ1 or 300 ng/mL IFNλ1-3. 24 h post treatment, cells were infected with Vaccinia virus-eGFP (VV) or Mammalian Reovirus (MRV) at an MOI of 1 (as determined in T84 WT cells). Infection media was supplemented with the respective IFN. 16 h post infection, cells were fixed, immunostained for viral protein and fluorescence imaging analysis was performed. ( A ) Schematic of the experimental setup. ( B ) Representative images showing Vaccinia virus eGFP (green) infected cells. Nuclei were stained with DAPI (blue). Scale bar = 200 µm. ( C , D ) Quantification of the number of ( C ) Vaccinia virus eGFP infected cells and ( D ) MRV infected cells. n = 3 biological replicates, error bars indicate the standard deviation. n.s. = not significant, P < 0.01 **, P < 0.001 ***, P < 0.0001 **** as determined by ordinary one-way ANOVA with Dunnett’s multiple comparison test. Testing was performed within high or low density groups, using only virus infected cells (no pretreatment) as reference. .

Article Snippet: Human recombinant IFNλ1 (IL-29) (#300-02L), IFNλ2 (IL28A) (#300-2K), and IFNλ3 (IL-28B) (#300-2K) were purchased from Peprotech, and cells were treated by a cocktail of all three type III IFNs in a ratio of 1:1:1, resulting in a final concentration of 300 ng/mL or as described in the figure legend.

Techniques: Infection, Virus, Fluorescence, Imaging, Staining, Standard Deviation, Comparison

Journal: bioRxiv

Article Title: Population context drives cell-to-cell variability in interferon response in epithelial cells

doi: 10.1101/2023.05.22.541682

Figure Lengend Snippet: T84-prom-Mx1-fp seeded at medium density were mock treated or treated with 2000 IU/mL IFNβ1 or 300 ng/mL IFNλ1-3 for 24 h. Cell nuclei were stained with DAPI and fluorescence microscopy was performed. (A) Representative images of cells (nuclei stained with DAPI are blue) expressing the fluorescent reporter (white). Yellow arrows point at IFN responder single cells or cells located at the colony edges. Red arrows point at non-responder cells in the colony center. (B) Correlation between single cell location and IFN-responsiveness was assessed using DBSCAN-CellX. Schematics depicting how the tool annotates cells according to their location at the edge or at the center of a cluster, or according to their edge degree are shown. (C, D) Quantification of the percentage of positive fluorescent cells as compared to mock-treated cells. (C) Edge vs. center cells. (D) Percentage of positive fluorescent cells dependent on the edge degree. An edge degree of 0 define single cells (no neighbors) and edge degree 1 are cells at the border of a colony. The higher the edge degree, the larger the distance from the edge. Error bars indicate standard deviations. n ≥ 3 biological replicates. n.s. =not significant. P<0.05 *, P<0.01 **, P<0.001 ***, P <0.0001 **** as determined by (C) Unpaired t test with Welch’s correction, and (D) ordinary one-way ANOVA with Dunnett’s multiple comparison test using edge degree 1 as reference.

Article Snippet: Human recombinant IFNλ1 (IL-29) (#300-02L), IFNλ2 (IL28A) (#300-2K) and IFNλ3 (IL-28B) (#300-2K) were purchased from Peprotech, and cells were treated by a cocktail of all three type III IFNs in a ratio of 1:1:1, resulting in a final concentration of 300 ng/mL or as described in the figure legend.

Techniques: Staining, Fluorescence, Microscopy, Expressing

Journal: bioRxiv

Article Title: Population context drives cell-to-cell variability in interferon response in epithelial cells

doi: 10.1101/2023.05.22.541682

Figure Lengend Snippet: (A) Schematic depicting the glass micropatterning approach using a Quartz-mask. (B, C) T84-prom-Mx1-fp cells seeded on circular micropatterns (200 µm diameter) were mock treated or treated with 2000 IU/mL IFNβ1 or 300 ng/mL IFNλ1-3. Fluorescent imaging was performed at 0 h, 12 h, and 24 h post treatment. (B) Representative images. The red line represents the edge of the patterns. Expression of the fluorescent reporter is depicted in white. Scale bar = 100µm. (C) The reporter expression for each single population was quantified by measuring the mean fluorescence intensity (MFI) at the edge and the center of a population at 12 h or 24 h post treatment, and normalizing it to the corresponding population 0 h post treatment. Each dot is one cell population (seeded on one micropattern), lines connect edge and center of the same cell population. n ≥ 3 biological replicates. n.s. =not significant, P <0.0001 **** as determined by Paired t test.

Article Snippet: Human recombinant IFNλ1 (IL-29) (#300-02L), IFNλ2 (IL28A) (#300-2K) and IFNλ3 (IL-28B) (#300-2K) were purchased from Peprotech, and cells were treated by a cocktail of all three type III IFNs in a ratio of 1:1:1, resulting in a final concentration of 300 ng/mL or as described in the figure legend.

Techniques: Imaging, Expressing, Fluorescence

Journal: bioRxiv

Article Title: Population context drives cell-to-cell variability in interferon response in epithelial cells

doi: 10.1101/2023.05.22.541682

Figure Lengend Snippet: T84 cells seeded at high and low density were mock treated, or treated with 2000 IU/mL IFNβ1 or 300 ng/mL IFNλ1-3. (A) Schematic depicting the experimental setup. (B) 24 h post IFN treatment, RNA was harvested to evaluate the transcription of the representative ISGs IFIT1, Mx1, and Viperin using q-RT-PCR. ISG relative expression was normalized to the mock-treated cells of the respective time-point (fold change). (C) 1 h post treatment, cellular protein extracts were collected to assess the phospho-STAT1 (pSTAT1) abundance by Western Blot. pSTAT1 was quantified relative to the housekeeping protein α-tubulin. (B, C) n = 3 biological replicates. n.s. =not significant. P<0.05 *, P<0.01 **, P<0.001 ***, P <0.0001 **** as determined by Unpaired t test with Welch’s correction.

Article Snippet: Human recombinant IFNλ1 (IL-29) (#300-02L), IFNλ2 (IL28A) (#300-2K) and IFNλ3 (IL-28B) (#300-2K) were purchased from Peprotech, and cells were treated by a cocktail of all three type III IFNs in a ratio of 1:1:1, resulting in a final concentration of 300 ng/mL or as described in the figure legend.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot

Journal: bioRxiv

Article Title: Population context drives cell-to-cell variability in interferon response in epithelial cells

doi: 10.1101/2023.05.22.541682

Figure Lengend Snippet: (A) T84 cells seeded on transwell inserts were mock treated, or treated from the apical (A) or basolateral (BL) side with 2000 IU/mL IFNβ1 or 300 ng/mL IFNλ1-3. 24 h post treatment, RNA was harvested, and q-RT-PCR was used to evaluate the expression of the ISG IFIT1. Data is normalized to mock (fold change). (B, C) T84 prom-Mx1-fp cells were seeded on micropatterned transwell membranes. Cells were mock treated, or treated simultaneously from the apical and basolateral side with 2000 IU/mL IFNβ1 or 300 ng/mL IFNλ1-3. Cells were fixed at 0 h, 12 h, and 24 h post treatment and fluorescent imaging was performed. (B) Representatives images showing treated T84 cell populations. The red line represents the edge of the patterns. Expression of the fluorescent reporter is depicted in white. Scale bar=100µm. (C) The reporter expression was quantified by measuring the mean fluorescence intensity (MFI) at the edge and the center of a population at 12 h or 24 h post treatment, and normalizing it to the corresponding 0 h post treatment at the edge and center, respectively. Each dot is one cell population (seeded on one micropattern), lines connect edge and center of the same cell population. (A, C) n ≥ 3 biological replicates. n.s. =not significant, P<0.01 **, P<0.001 *** as determined by (A) Unpaired t test with Welch’s correction, and (C) Paired t test.

Article Snippet: Human recombinant IFNλ1 (IL-29) (#300-02L), IFNλ2 (IL28A) (#300-2K) and IFNλ3 (IL-28B) (#300-2K) were purchased from Peprotech, and cells were treated by a cocktail of all three type III IFNs in a ratio of 1:1:1, resulting in a final concentration of 300 ng/mL or as described in the figure legend.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Imaging, Fluorescence

Journal: bioRxiv

Article Title: Population context drives cell-to-cell variability in interferon response in epithelial cells

doi: 10.1101/2023.05.22.541682

Figure Lengend Snippet: (A) Schematic depicting paracellular diffusion in a monolayer of T84 WT and T84 ZO1 KO cells with disrupted junctional complexes. (B) T84 WT and T84 ZO1 KO cell protein extracts were harvested to control the absence of ZO1 protein in the KO cells by Western Blot. α-tubulin served as a housekeeping protein. Representative image is shown. (C) T84 WT and T84 ZO1 KO cells were fixed and indirect immunofluorescence was performed against the junctional complex protein ZO1 (green). Nuclei were stained with DAPI (blue). Representative image is shown. (D) T84 WT and T84 ZO1 KO cells were seeded on transwell inserts and grown as a polarized monolayer. Transepithelial electrical resistance (TEER) was measured over a period of 5 days. Dotted line shows a TEER of 1000 Ω/cm 2 corresponding to the resistance formed by confluent polarized T84 cells . (E) T84 WT and T84 ZO1 KO cells at high (H) and low (L) density were treated apically with 2000 IU/mL IFNβ1 or 300 ng/mL IFNλ1-3. 24 h post treatment, RNA was harvested, and q-RT-PCR was used to evaluate the expression of the ISG IFIT1. Data is normalized to mock (fold change). (D, E) n ≥ 3 biological replicates. n.s. = not significant, P<0. 05 *, P<0.01 **, P<0.001 ***, P <0.0001 **** as determined by (D) ordinary one-way ANOVA with Dunnett’s multiple comparison test using the positive control as reference, and (E) Unpaired t test with Welch’s correction.

Article Snippet: Human recombinant IFNλ1 (IL-29) (#300-02L), IFNλ2 (IL28A) (#300-2K) and IFNλ3 (IL-28B) (#300-2K) were purchased from Peprotech, and cells were treated by a cocktail of all three type III IFNs in a ratio of 1:1:1, resulting in a final concentration of 300 ng/mL or as described in the figure legend.

Techniques: Diffusion-based Assay, Western Blot, Immunofluorescence, Staining, Reverse Transcription Polymerase Chain Reaction, Expressing, Positive Control

Journal: bioRxiv

Article Title: Population context drives cell-to-cell variability in interferon response in epithelial cells

doi: 10.1101/2023.05.22.541682

Figure Lengend Snippet: T84 cells seeded at high and low density were mock-treated or pre-treated with 2000 IU/mL IFNβ1 or 300 ng/mL IFNλ1-3. 24 h post treatment, cells were infected with Vaccinia virus-eGFP (VV) or Mammalian Reovirus (MRV) at an MOI of 1 (as determined in T84 WT cells). Infection media was supplemented with the respective IFN. 16 h post infection, cells were fixed, immunostained for viral protein and fluorescence imaging analysis was performed. (A) Schematic of the experimental setup. (B) Representative images showing Vaccinia virus eGFP (green) infected cells. Nuclei were stained with DAPI (blue). Scale bar=200µm. (C, D) Quantification of the number of (C) Vaccinia virus eGFP infected cells and (D) MRV infected cells. n = 3 biological replicates. n.s. = not significant, P<0.01 **, P<0.001 ***, P <0.0001 **** as determined by ordinary one-way ANOVA with Dunnett’s multiple comparison test. Test was performed within high or low density groups, using only virus infected cells (no pretreatment) as reference.

Article Snippet: Human recombinant IFNλ1 (IL-29) (#300-02L), IFNλ2 (IL28A) (#300-2K) and IFNλ3 (IL-28B) (#300-2K) were purchased from Peprotech, and cells were treated by a cocktail of all three type III IFNs in a ratio of 1:1:1, resulting in a final concentration of 300 ng/mL or as described in the figure legend.

Techniques: Infection, Fluorescence, Imaging, Staining