Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Silver nanoparticles at sub-cytotoxic levels increase enteric pathogen invasion by compromising intestinal epithelial barrier integrity

doi: 10.3389/fcimb.2026.1745955

Figure Lengend Snippet: Effect of 10 and 20 µg/mL AgNPs pretreatment on bacterial adhesion, invasion, and persistence in infected T84 epithelial cells. T84 cells were pretreated or 24 hours with 10 nm AgNPs at 10 or 20 µg/mL, followed infection with Salmonella enterica serovar Heidelberg strain 146. (a) Bacterial adhesion, invasion, and persistence were quantified by measuring CFU/mL (as described in M&Ms). (b) Bacterial survival percentage in T84 cells during adhesion, invasion and persistence following AgNPs treatment. Survival was calculated by dividing CFU/mL (as described in M&Ms) for each condition by its corresponding control (set to 100% and therefore not shown as a separate bar) and expressed as a percentage. Bars represent mean ± SD from three independent experiments performed in biological triplicates. Asterisks indicate statistically significant differences compared to respective untreated infection controls (** p < 0.005, *** p < 0.0005; unpaired t-test).

Article Snippet: Human colon carcinoma T84 epithelial cells (ATCC ® CCL-248TM) were cultured in complete medium composed of Dulbecco’s Modified Eagle Medium/Ham’s F-12 (DMEM/F-12) supplemented with l-glutamine and HEPES (ATCC, Manassas, VA, USA), 10% fetal bovine serum (FBS), 1% penicillin-streptomycin and 0.1% fungizone (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Infection, Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Silver nanoparticles at sub-cytotoxic levels increase enteric pathogen invasion by compromising intestinal epithelial barrier integrity

doi: 10.3389/fcimb.2026.1745955

Figure Lengend Snippet: Relative expression of genes related to cell-cell junction and epithelial barrier function in T84 cells following AgNPs exposure and bacterial infection. T84 cells were pretreated with 10 nm AgNPs at either 10 µg/mL or 20 µg/mL, alone or in combination with S. enterica under invasion (1 h + gentamicin) or persistence (24 h + gentamicin) conditions. Invasion control consists of infected cells that are not pretreated with AgNPs but treated with gentamicin for 1 hour, while persistence control consists of infected cells not pretreated with AgNPs but treated with gentamicin for 24 hours. Gene expression (as described in M&Ms) was measured using RT² Profiler PCR arrays and is represented as Log 2 fold regulation relative to untreated control cells. (a) Focal adhesion genes, (b) Gap junction genes, (c) Tight junction genes, and (d) Adherens Junctions, desmosomes, and hemidesmosomes genes. Bars represent mean ± SE from biological triplicates. Asterisks indicate statistically significant differences compared to uninfected and AgNPs untreated control (p < 0.05) (*p < 0.05, **p < 0.005; unpaired t-test).

Article Snippet: Human colon carcinoma T84 epithelial cells (ATCC ® CCL-248TM) were cultured in complete medium composed of Dulbecco’s Modified Eagle Medium/Ham’s F-12 (DMEM/F-12) supplemented with l-glutamine and HEPES (ATCC, Manassas, VA, USA), 10% fetal bovine serum (FBS), 1% penicillin-streptomycin and 0.1% fungizone (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing, Infection, Control, Gene Expression

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Silver nanoparticles at sub-cytotoxic levels increase enteric pathogen invasion by compromising intestinal epithelial barrier integrity

doi: 10.3389/fcimb.2026.1745955

Figure Lengend Snippet: Concentration of pro- and anti-inflammatory cytokines in T84 epithelial cells following AgNPs exposure and bacterial infection. Cytokine concentrations in the supernatants of T84 cells pretreated with 10 µg/mL or 20 µg/mL of 10 nm AgNPs and exposed to S. enterica under invasion or persistence conditions. (a) Pro-inflammatory cytokines and (b) Anti-inflammatory cytokine. Bars represent mean ± SE from biological triplicates. Asterisks indicate statistically significant differences compared to uninfected and AgNPs untreated control (p < 0.05).

Article Snippet: Human colon carcinoma T84 epithelial cells (ATCC ® CCL-248TM) were cultured in complete medium composed of Dulbecco’s Modified Eagle Medium/Ham’s F-12 (DMEM/F-12) supplemented with l-glutamine and HEPES (ATCC, Manassas, VA, USA), 10% fetal bovine serum (FBS), 1% penicillin-streptomycin and 0.1% fungizone (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Concentration Assay, Infection, Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Silver nanoparticles at sub-cytotoxic levels increase enteric pathogen invasion by compromising intestinal epithelial barrier integrity

doi: 10.3389/fcimb.2026.1745955

Figure Lengend Snippet: Concentration of growth factors, chemokines, and Th2 cytokines in T84 epithelial cells following AgNPs exposure and bacterial infection. T84 cells were treated with 10 nm AgNPs at 10 µg/mL or 20 µg/mL, either alone or in combination with S. enterica invasion or persistence. Invasion control consisted of infected cells that were not pretreated with AgNPs but treated with gentamicin for 1 hour, while persistence control consisted of infected cells not pretreated with AgNPs but treated with gentamicin for 24 hours. (a) Growth factors, (b) chemokines, and (c) Th2 cytokines. Bars represent mean ± SE from biological triplicates. Asterisks indicate statistically significant differences compared to control (p < 0.05).

Article Snippet: Human colon carcinoma T84 epithelial cells (ATCC ® CCL-248TM) were cultured in complete medium composed of Dulbecco’s Modified Eagle Medium/Ham’s F-12 (DMEM/F-12) supplemented with l-glutamine and HEPES (ATCC, Manassas, VA, USA), 10% fetal bovine serum (FBS), 1% penicillin-streptomycin and 0.1% fungizone (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Concentration Assay, Infection, Control

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

Journal: Molecular Therapy Oncology

Article Title: Tackling cancer heterogeneity with systemically delivered oncolytic adenoviruses transcriptionally targeted with hybrid promoters

doi: 10.1016/j.omton.2025.201073

Figure Lengend Snippet: mRNA and protein expression levels of selected candidates in human CRC cell lines and patients’ samples (A–F) mRNA expression levels of the six selected candidates for inclusion in the hybrid TSP. Quantitative PCR (qPCR) analysis was performed using specific primers for each candidate gene (vWA2, A33, FSCN1, NQO1, PDL1, and AQP5) in four human colorectal cancer cell lines (T84, HCT116, HT29, and LoVo) and in normal human colonic epithelial cells (CCD841). qPCR data are presented as relative quantification (RQ = 2 −ΔΔCt ). Statistical analysis was performed using one-way ANOVA. Data represent the mean of three independent experiments. (G) Immunohistochemical analysis of gpA33 and vWA2 expression was performed on paraffin-embedded primary and metastatic CRC tissue samples. (a and b) Representative images of primary tumor sections stained with an anti-gpA33 antibody and developed as described in the . (c and d) Representative images of primary tumor sections stained with an anti-vWA2 antibody under the same conditions. (e–g) Representative images of uterine, cutaneous, and liver metastases, respectively, stained with the anti-gpA33 antibody. (h–j) Corresponding metastatic tissue sections (uterus, skin, and liver, respectively) stained with an anti-vWA2 antibody. All images were acquired at an original magnification of 200×. Scale bar: 50 μm.

Article Snippet: Human CRC cell lines (LoVo, T84, HCT116, and HT29), normal colon epithelial cells (CCD841), human embryonic kidney cells (HEK293), human fetal lung fibroblasts (WI-38), and human microendothelial cells (HMEC-1) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Quantitative Proteomics, Immunohistochemical staining, Staining

Journal: Molecular Therapy Oncology

Article Title: Tackling cancer heterogeneity with systemically delivered oncolytic adenoviruses transcriptionally targeted with hybrid promoters

doi: 10.1016/j.omton.2025.201073

Figure Lengend Snippet: The hybrid TSP vWA33 exhibits transcriptional activity across all human CRC cell lines tested (A and C) Identification of transcription factor (TF) binding motifs within the vWA2 (A) and A33 (C) promoter fragments. A comprehensive in silico analysis was performed on the six candidate promoter fragments (only vWA2 and A33 are shown). TFs and their positions were identified using a combination of PROMO, MATCH, and MEME Suite – FIMO and filtered for consensus hits across all three tools. ToppGene was used to rank the most statistically significant TFs, as described in the . (B) Transcriptional activity of the full-length vWA2 promoter (636 bp) vs. the shorter, transcriptionally silent 306 bp fragment lacking RNA polymerase binding motifs. Both fragments were cloned into a promoter-less luciferase vector and transfected into four CRC cell lines. Luciferase activity was used as a readout and is presented as a fold induction relative to the pGL3-Basic control. (D) Schematic representation of the vWA33 hybrid promoter, composed of the vWA2 fragment (−325 to −19 bp) followed by the A33 promoter fragment (−105 to +307 bp). Annotated TF binding sites are indicated. (E) Comparative transcriptional activity of the A33 promoter (Pr A33), vWA2 promoter (Pr vWA2), and vWA33 hybrid promoter (Pr vWA33) in four CRC cell lines, using luciferase as the reporter. The data shown represent one representative experiment out of at least three independent replicates. One-way ANOVA was used for statistical analysis.

Article Snippet: Human CRC cell lines (LoVo, T84, HCT116, and HT29), normal colon epithelial cells (CCD841), human embryonic kidney cells (HEK293), human fetal lung fibroblasts (WI-38), and human microendothelial cells (HMEC-1) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Activity Assay, Binding Assay, In Silico, Clone Assay, Luciferase, Plasmid Preparation, Transfection, Control

Journal: Molecular Therapy Oncology

Article Title: Tackling cancer heterogeneity with systemically delivered oncolytic adenoviruses transcriptionally targeted with hybrid promoters

doi: 10.1016/j.omton.2025.201073

Figure Lengend Snippet: AR2015 lytic activity in vitro compared to single TSP-driven OAds (A–I) In vitro lytic activity of the OAds AR2015, AV22EL, and AV636, as well as Ad5WT (positive control), in CRC cell lines LoVo, T84, HT29, and HCT116, normal human colonic epithelial cells (CCD841), human fetal lung fibroblasts (WI-38), human microendothelial cells (HMEC-1), and human melanoma cells (A375 and SB2). Cells (1 × 10 4 ) were seeded in 24-well plates and infected 24 h later with increasing multiplicities of infection (MOIs: 0–100). After 6 days, cell viability was assessed using the MTS assay and expressed as mean ± SD ( n = 3), with the viability of uninfected control cells set to 100%. Two-way ANOVA was performed for statistical analysis, followed by Dunnett’s test (vs. Ad(F5)WT). (J) Comparative replication kinetics of AR2015 and AV22EL in CRC cell lines (LoVo, T84, HT29, and HCT116) and normal CCD841 cells. Cells were infected at an MOI of 100, and samples were collected at 5 h (baseline) and 72 h post-infection. Viral replication was quantified by qPCR targeting the adenoviral E4 gene. Data are presented as a fold increase in E4 levels (72 vs. 5 h). (K) Expression of adenoviral E1A protein in LoVo cells at 8 and 24 h post-infection with AV22EL, AR2015, or Ad5WT. Protein levels were assessed by western blot, with β-actin used as a loading control.

Article Snippet: Human CRC cell lines (LoVo, T84, HCT116, and HT29), normal colon epithelial cells (CCD841), human embryonic kidney cells (HEK293), human fetal lung fibroblasts (WI-38), and human microendothelial cells (HMEC-1) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Activity Assay, In Vitro, Positive Control, Infection, MTS Assay, Control, Expressing, Western Blot

Journal: Molecular Therapy Oncology

Article Title: Tackling cancer heterogeneity with systemically delivered oncolytic adenoviruses transcriptionally targeted with hybrid promoters

doi: 10.1016/j.omton.2025.201073

Figure Lengend Snippet: Systemically administered AR2015 exhibits superior efficacy against established liver metastases compared to single TSP-driven OAds (A) Schematic diagram of the experimental protocol. Nude mice were injected intrasplenically with 2 × 10 6 HCT116 CRC cells, and 15 days later, when liver metastases were established, mice were systemically treated with the indicated OAds (5 × 10 9 viral particles/mouse) with or without a suboptimal dose of 5-FU (30 mg/kg). (B) Kaplan-Meier survival plot of mice bearing HCT116 liver metastases treated with PBS ( n = 6), 5-FU ( n = 6), AV22EL ( n = 3), AR2015 ( n = 7), or AR2015 + 5-FU ( n = 6). Statistical significance was calculated using the log rank test. Mice were euthanized when the tumor burden reached 2 cm 3 , in accordance with ethical guidelines. (C and D) Presence (C) and quantification of area (D) of metastatic liver foci from the experimental groups in (B). (E and F) In an independent experiment, nude mice were injected intrasplenically with 2 × 10 6 HCT116 cells, and 15 days later, they were treated with PBS ( n = 6), AV636 ( n = 6), or AR2015 ( n = 6). Mice were euthanized at the endpoint and analyzed for the presence (E) and area (F) of hepatic metastatic lesions. One-way ANOVA was used for statistical analysis of metastatic burden.

Article Snippet: Human CRC cell lines (LoVo, T84, HCT116, and HT29), normal colon epithelial cells (CCD841), human embryonic kidney cells (HEK293), human fetal lung fibroblasts (WI-38), and human microendothelial cells (HMEC-1) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Injection

Journal: Molecular Therapy Oncology

Article Title: Tackling cancer heterogeneity with systemically delivered oncolytic adenoviruses transcriptionally targeted with hybrid promoters

doi: 10.1016/j.omton.2025.201073

Figure Lengend Snippet: Systemically delivered AR2015 demonstrates enhanced in vivo efficacy in combination with oxaliplatin and strong in vitro lytic activity in patient-derived xenograft CRC cells (A and B) Nude mice were injected intrasplenically with 2 × 10 6 LoVo CRC cells. 15 days post-injection, mice were treated with PBS ( n = 4), oxaliplatin (OXA) ( n = 3), AR2015 ( n = 5), or AR2015 + OXA ( n = 5). At the experimental endpoint, mice were euthanized and assessed for the presence of liver metastases (A) and metastatic lesion area (B). (C and F) qPCR analysis of vWA2, A33, CAR, and DSG-2 mRNA expression in RC-01 and RC-04 patient-derived xenograft (PDX)-derived CRC cells. Expression is presented as relative quantification (RQ = 2 −ΔΔCt ). (D and G) In vitro lytic activity of AR2015 and AR2015(F5/3) in RC-01 and RC-04 cells. Cells (1 × 10 4 per well) were seeded in 24-well plates and infected 24 h later with increasing MOIs (0–100). Cell viability was assessed after 6 days using the MTS assay. (E and H) Replication capacity of AR2015 and AR2015(F5/3) in RC-01 and RC-04 cells. Cells (1 × 10 4 ) were infected at an MOI of 100, and samples were collected at 5 (baseline) and 72 h. Viral replication was assessed by qPCR targeting the adenoviral E4 gene. Data are presented as a fold increase in E4 levels (72 vs. 5 h). One-way ANOVA was used for statistical analysis. p values are indicated where applicable.

Article Snippet: Human CRC cell lines (LoVo, T84, HCT116, and HT29), normal colon epithelial cells (CCD841), human embryonic kidney cells (HEK293), human fetal lung fibroblasts (WI-38), and human microendothelial cells (HMEC-1) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: In Vivo, In Vitro, Activity Assay, Derivative Assay, Injection, Expressing, Quantitative Proteomics, Infection, MTS Assay