Journal: Molecular Therapy Oncology

Article Title: ZEB1, a novel junctional adhesion molecule A regulator, impacts sensitivity of pancreatic cancer-associated fibroblasts to reovirus

doi: 10.1016/j.omton.2025.201071

Figure Lengend Snippet: ZEB1 KD, but not FGFR1 KD, results in increased JAM-A expression in JAM-A-positive and negative human and murine fibroblast cell lines and sensitization to reovirus (A–D) RT-qPCR for ZEB1 expression in RLT-PSC (A) and KPC3-CAF1 (C) vector control and ZEB1 KD and FGFR1 expression in RLT-PSC (B) and KPC3-CAF1 (D) vector control and FGFR1 KD. Ct values were corrected for IPO8 and EIF2B1 (RLT-PSC) or Mzt2 and Ptp4a (KPC3-CAF1) expression and calculated as fold change vs. vector control. Significance was calculated using one-way ANOVA with correction for multiple testing (Šídák’s test) or unpaired t test for KPC3-CAF1 FGFR1 KD, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001. Data are derived from a representative experiment and plotted as mean ± SD. (E and F) Western blot for ZEB1 (200 kDa) with vinculin (117 kDa) as loading control in RLT-PSC (E) and KPC3-CAF1 (F) vector control and ZEB1 KD. Asterisk indicates a specific band. Band intensity was determined using Image Studio Lite software and calculated as fold change versus the WT cell line. (G and H) Flow cytometric analysis of cell-surface JAM-A expression in RLT-PSC (G) and KPC3-CAF1 (H) vector control, FGFR1 KD, and ZEB1 KD. Black: secondary antibody only, blue: stained. (I and J) Cell viability (%) relative to mock following infection of RLT-PSC (I) and KPC3-CAF1 (J) vector control and FGFR1 and ZEB1 KDs with reovirus at multiple MOIs for 3 days, as measured by a WST-1 assay. Significance was calculated using two-way ANOVA with correction for multiple testing (Šídák’s test), significance is depicted at MOI 10, ∗∗∗∗ p ≤ 0.0001. Data are derived from a representative experiment and plotted as mean ± SD.

Article Snippet: For chromatin immunoprecipitation assays, hPS1 ZEB1 KO cells, rescued with ZEB1 cDNA (clone 2D12+ZEB1), were cultured for 48 h. One day prior to fixation of the cells, Protein A dynabeads (Thermo Fisher Scientific) were coated with 4 μg ZEB1 (Proteintech 21544-1-AP, Manchester, UK) or 4 μg rabbit IgG control Ab (PP64, Sigma-Aldrich) overnight in PBS/1% BSA (>98% BSA free, Sigma-Aldrich).

Techniques: Expressing, Quantitative RT-PCR, Plasmid Preparation, Control, Derivative Assay, Western Blot, Software, Staining, Infection, WST-1 Assay

Journal: Molecular Therapy Oncology

Article Title: ZEB1, a novel junctional adhesion molecule A regulator, impacts sensitivity of pancreatic cancer-associated fibroblasts to reovirus

doi: 10.1016/j.omton.2025.201071

Figure Lengend Snippet: ZEB1 KO in human and murine pancreatic fibroblasts results in increased JAM-A RNA and protein through direct transcriptional regulation (A–C) Western blot of clonal ZEB1 KO and ZEB1 rescues (200 kDa) in RLT-PSC (A), hPS1 (B), and KPC3-CAF1 (C) fibroblasts with vinculin (117 kDa) as loading control. Asterisk indicates a specific band. (D–F) RT-qPCR analysis of F11R expression in ZEB1 KO and rescue cell lines RLT-PSC (D), hPS1 (E), and KPC3-CAF1 (F). Ct values were corrected for IPO8 and EIF2B1 (RLT-PSC and hPS1) or MZT2 and PTP4A2 (KPC3-CAF1) expression and calculated as fold change vs. WT. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗∗ p ≤ 0.0001 as determined by one-way ANOVA with correction for multiple testing (Šídák’s test) Data are derived from a representative experiment and plotted as mean ± SD. (G–I) Flow cytometric analyses of cell-surface JAM-A expression of ZEB1 KO clones and rescues in RLT-PSC (G), hPS1 (H), and KPC3-CAF1 (I). Black: secondary only antibody, red: stained. (J–L) Geometric mean fluorescent intensity (gMFI) of JAM-A expression of the different cell lines as depicted in (G), (H), and (I). ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001 as determined by one-way ANOVA with correction for multiple testing (Šídák’s test). Data are derived from a representative experiment and plotted as mean ± SD. (M) Chromatin immunoprecipitation qPCR (ChIP-qPCR) assay to identify DNA binding regions of ZEB1. 1–4: different E-box binding regions within the F11R promoter, CDH1: E-box binding region within the E-cadherin promoter. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗∗ p ≤ 0.0001 as determined by two-way ANOVA with correction for multiple testing (Šídák’s test). Data are derived from a representative experiment and plotted as mean ± SD.

Article Snippet: For chromatin immunoprecipitation assays, hPS1 ZEB1 KO cells, rescued with ZEB1 cDNA (clone 2D12+ZEB1), were cultured for 48 h. One day prior to fixation of the cells, Protein A dynabeads (Thermo Fisher Scientific) were coated with 4 μg ZEB1 (Proteintech 21544-1-AP, Manchester, UK) or 4 μg rabbit IgG control Ab (PP64, Sigma-Aldrich) overnight in PBS/1% BSA (>98% BSA free, Sigma-Aldrich).

Techniques: Western Blot, Control, Quantitative RT-PCR, Expressing, Derivative Assay, Clone Assay, Staining, Chromatin Immunoprecipitation, ChIP-qPCR, Binding Assay

Journal: Molecular Therapy Oncology

Article Title: ZEB1, a novel junctional adhesion molecule A regulator, impacts sensitivity of pancreatic cancer-associated fibroblasts to reovirus

doi: 10.1016/j.omton.2025.201071

Figure Lengend Snippet: ZEB1 KO in human and murine fibroblasts results in increased susceptibility to reovirus infection and reovirus-mediated apoptotic cell death (A, E, and I) Cell viability (%) relative to mock following infection with reovirus at multiple MOIs, as measured by a WST-1 assay. RLT-PSC (A) were infected for 4 days, hPS1 (E) for 6 days, and KPC3-CAF1 (I) for 3 days. Significance was calculated using two-way ANOVA with correction for multiple testing (Šídák’s test), significance is depicted at MOI 10, ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗∗ p ≤ 0.0001. Data are derived from a representative experiment and plotted as mean ± SD. (B, F, and J) Overlay of phase-contrast and GFP images of CellEvent Caspase 3/7 assay of RLT-PSC (B), hPS1 (F), and KPC3-CAF1 (J) WT and ZEB1 KO cells. Cells were infected with R124 MOI 10 for 36, 48, and 24 h, respectively. Scale bars: 200 μM. (C, G, and K) Quantification of CellEvent Caspase 3/7 fluorescent signal over time, during infection with reovirus R124 MOI 10 for 36 (RLT-PSC, C), 48 (hPS1, G), or 24 h (KPC3-CAF1, K). Significance was calculated using two-way ANOVA with correction for multiple testing (Šídák’s test), ∗∗∗∗ p ≤ 0.0001. Data are derived from a representative experiment and plotted as mean ± SD. (D, H, and L) Western blot for reovirus Sigma3 expression (41 kDa) and vinculin (117 kDa) as loading control in WT and ZEB1 KO cells following reovirus infection at different MOIs. RLT-PSC (B) and hPS1 (E) were infected for 2 days and KPC3-CAF1 (H) for 1 day.

Article Snippet: For chromatin immunoprecipitation assays, hPS1 ZEB1 KO cells, rescued with ZEB1 cDNA (clone 2D12+ZEB1), were cultured for 48 h. One day prior to fixation of the cells, Protein A dynabeads (Thermo Fisher Scientific) were coated with 4 μg ZEB1 (Proteintech 21544-1-AP, Manchester, UK) or 4 μg rabbit IgG control Ab (PP64, Sigma-Aldrich) overnight in PBS/1% BSA (>98% BSA free, Sigma-Aldrich).

Techniques: Infection, WST-1 Assay, Derivative Assay, Western Blot, Expressing, Control

Journal: Molecular Therapy Oncology

Article Title: ZEB1, a novel junctional adhesion molecule A regulator, impacts sensitivity of pancreatic cancer-associated fibroblasts to reovirus

doi: 10.1016/j.omton.2025.201071

Figure Lengend Snippet: ZEB1 KD, but not FGFR1 KD, results in increased JAM-A expression in JAM-A-positive and negative human and murine fibroblast cell lines and sensitization to reovirus (A–D) RT-qPCR for ZEB1 expression in RLT-PSC (A) and KPC3-CAF1 (C) vector control and ZEB1 KD and FGFR1 expression in RLT-PSC (B) and KPC3-CAF1 (D) vector control and FGFR1 KD. Ct values were corrected for IPO8 and EIF2B1 (RLT-PSC) or Mzt2 and Ptp4a (KPC3-CAF1) expression and calculated as fold change vs. vector control. Significance was calculated using one-way ANOVA with correction for multiple testing (Šídák’s test) or unpaired t test for KPC3-CAF1 FGFR1 KD, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001. Data are derived from a representative experiment and plotted as mean ± SD. (E and F) Western blot for ZEB1 (200 kDa) with vinculin (117 kDa) as loading control in RLT-PSC (E) and KPC3-CAF1 (F) vector control and ZEB1 KD. Asterisk indicates a specific band. Band intensity was determined using Image Studio Lite software and calculated as fold change versus the WT cell line. (G and H) Flow cytometric analysis of cell-surface JAM-A expression in RLT-PSC (G) and KPC3-CAF1 (H) vector control, FGFR1 KD, and ZEB1 KD. Black: secondary antibody only, blue: stained. (I and J) Cell viability (%) relative to mock following infection of RLT-PSC (I) and KPC3-CAF1 (J) vector control and FGFR1 and ZEB1 KDs with reovirus at multiple MOIs for 3 days, as measured by a WST-1 assay. Significance was calculated using two-way ANOVA with correction for multiple testing (Šídák’s test), significance is depicted at MOI 10, ∗∗∗∗ p ≤ 0.0001. Data are derived from a representative experiment and plotted as mean ± SD.

Article Snippet: For chromatin immunoprecipitation assays, hPS1 ZEB1 KO cells, rescued with ZEB1 cDNA (clone 2D12+ZEB1), were cultured for 48 h. One day prior to fixation of the cells, Protein A dynabeads (Thermo Fisher Scientific) were coated with 4 μg ZEB1 (Proteintech 21544-1-AP, Manchester, UK) or 4 μg rabbit IgG control Ab (PP64, Sigma-Aldrich) overnight in PBS/1% BSA (>98% BSA free, Sigma-Aldrich).

Techniques: Expressing, Quantitative RT-PCR, Plasmid Preparation, Control, Derivative Assay, Western Blot, Software, Staining, Infection, WST-1 Assay

Journal: Molecular Therapy Oncology

Article Title: ZEB1, a novel junctional adhesion molecule A regulator, impacts sensitivity of pancreatic cancer-associated fibroblasts to reovirus

doi: 10.1016/j.omton.2025.201071

Figure Lengend Snippet: ZEB1 KO in human and murine pancreatic fibroblasts results in increased JAM-A RNA and protein through direct transcriptional regulation (A–C) Western blot of clonal ZEB1 KO and ZEB1 rescues (200 kDa) in RLT-PSC (A), hPS1 (B), and KPC3-CAF1 (C) fibroblasts with vinculin (117 kDa) as loading control. Asterisk indicates a specific band. (D–F) RT-qPCR analysis of F11R expression in ZEB1 KO and rescue cell lines RLT-PSC (D), hPS1 (E), and KPC3-CAF1 (F). Ct values were corrected for IPO8 and EIF2B1 (RLT-PSC and hPS1) or MZT2 and PTP4A2 (KPC3-CAF1) expression and calculated as fold change vs. WT. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗∗ p ≤ 0.0001 as determined by one-way ANOVA with correction for multiple testing (Šídák’s test) Data are derived from a representative experiment and plotted as mean ± SD. (G–I) Flow cytometric analyses of cell-surface JAM-A expression of ZEB1 KO clones and rescues in RLT-PSC (G), hPS1 (H), and KPC3-CAF1 (I). Black: secondary only antibody, red: stained. (J–L) Geometric mean fluorescent intensity (gMFI) of JAM-A expression of the different cell lines as depicted in (G), (H), and (I). ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001 as determined by one-way ANOVA with correction for multiple testing (Šídák’s test). Data are derived from a representative experiment and plotted as mean ± SD. (M) Chromatin immunoprecipitation qPCR (ChIP-qPCR) assay to identify DNA binding regions of ZEB1. 1–4: different E-box binding regions within the F11R promoter, CDH1: E-box binding region within the E-cadherin promoter. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗∗ p ≤ 0.0001 as determined by two-way ANOVA with correction for multiple testing (Šídák’s test). Data are derived from a representative experiment and plotted as mean ± SD.

Article Snippet: For chromatin immunoprecipitation assays, hPS1 ZEB1 KO cells, rescued with ZEB1 cDNA (clone 2D12+ZEB1), were cultured for 48 h. One day prior to fixation of the cells, Protein A dynabeads (Thermo Fisher Scientific) were coated with 4 μg ZEB1 (Proteintech 21544-1-AP, Manchester, UK) or 4 μg rabbit IgG control Ab (PP64, Sigma-Aldrich) overnight in PBS/1% BSA (>98% BSA free, Sigma-Aldrich).

Techniques: Western Blot, Control, Quantitative RT-PCR, Expressing, Derivative Assay, Clone Assay, Staining, Chromatin Immunoprecipitation, ChIP-qPCR, Binding Assay

Journal: Molecular Therapy Oncology

Article Title: ZEB1, a novel junctional adhesion molecule A regulator, impacts sensitivity of pancreatic cancer-associated fibroblasts to reovirus

doi: 10.1016/j.omton.2025.201071

Figure Lengend Snippet: ZEB1 KO in human and murine fibroblasts results in increased susceptibility to reovirus infection and reovirus-mediated apoptotic cell death (A, E, and I) Cell viability (%) relative to mock following infection with reovirus at multiple MOIs, as measured by a WST-1 assay. RLT-PSC (A) were infected for 4 days, hPS1 (E) for 6 days, and KPC3-CAF1 (I) for 3 days. Significance was calculated using two-way ANOVA with correction for multiple testing (Šídák’s test), significance is depicted at MOI 10, ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗∗ p ≤ 0.0001. Data are derived from a representative experiment and plotted as mean ± SD. (B, F, and J) Overlay of phase-contrast and GFP images of CellEvent Caspase 3/7 assay of RLT-PSC (B), hPS1 (F), and KPC3-CAF1 (J) WT and ZEB1 KO cells. Cells were infected with R124 MOI 10 for 36, 48, and 24 h, respectively. Scale bars: 200 μM. (C, G, and K) Quantification of CellEvent Caspase 3/7 fluorescent signal over time, during infection with reovirus R124 MOI 10 for 36 (RLT-PSC, C), 48 (hPS1, G), or 24 h (KPC3-CAF1, K). Significance was calculated using two-way ANOVA with correction for multiple testing (Šídák’s test), ∗∗∗∗ p ≤ 0.0001. Data are derived from a representative experiment and plotted as mean ± SD. (D, H, and L) Western blot for reovirus Sigma3 expression (41 kDa) and vinculin (117 kDa) as loading control in WT and ZEB1 KO cells following reovirus infection at different MOIs. RLT-PSC (B) and hPS1 (E) were infected for 2 days and KPC3-CAF1 (H) for 1 day.

Article Snippet: For chromatin immunoprecipitation assays, hPS1 ZEB1 KO cells, rescued with ZEB1 cDNA (clone 2D12+ZEB1), were cultured for 48 h. One day prior to fixation of the cells, Protein A dynabeads (Thermo Fisher Scientific) were coated with 4 μg ZEB1 (Proteintech 21544-1-AP, Manchester, UK) or 4 μg rabbit IgG control Ab (PP64, Sigma-Aldrich) overnight in PBS/1% BSA (>98% BSA free, Sigma-Aldrich).

Techniques: Infection, WST-1 Assay, Derivative Assay, Western Blot, Expressing, Control