Journal: iScience

Article Title: C9ORF72 suppresses JAK-STAT mediated inflammation

doi: 10.1016/j.isci.2023.106579

Figure Lengend Snippet:

Article Snippet: rabbit anti-pSTAT1 , R and D Systems , Cat# AF2894, RRID: AB_2198137.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Bradford Protein Assay, CRISPR, Plasmid Preparation, Software

Journal: Signal Transduction and Targeted Therapy

Article Title: Bioengineered iPSC-derived human macrophages with increased angiotensin-converting enzyme (ACE) expression suppress solid tumor growth

doi: 10.1038/s41392-026-02650-3

Figure Lengend Snippet: Determination of M1 vs. M2 polarization in iMac. a Pro-inflammatory M1 cytokines IL-1β, TNFα, IL-6, and IL-8 measured by ELISA. b Anti-inflammatory M2 cytokines IL-4, IL-10, IL-13, CCL5, CCL17, and CCL20 measured by ELISA. For a , b iMac were cultured in 6-well plates (3 × 10 6 cells/well). To challenge with tumor factors, iMac were co-cultured with 50% fresh supernatant from SK-MEL-28 cells every 24 h for 3 days. Supernatants were collected during the final 24 h for cytokine measurement. c Activation (phosphorylation) of NF-kB p65, STAT1, STAT3, and STAT6 in iMac was determined using Western blotting after conditioning with melanoma (SK-MEL-28) supernatant for 72 h. Protein band intensity was quantified using Odyssey version 3.0 software. After adjusting for loading using the β-actin band, data are presented as fold change to the iMac (Dox-) group, which is set as 1. For a – c , one-way ANOVA with Bonferroni’s correction for multiple comparisons was used to analyze group comparisons. Data are presented as means ± SD. Each experiment was performed three times, with triplicate samples each time. * p < 0.05, *** p < 0.001, and **** p < 0.0001

Article Snippet: The polyvinylidene difluoride (PVDF) membranes were incubated with specific primary antibodies against ACE (R&D Systems, MAB9291, 1:1,000), GAPDH (Sigma-Aldrich, SAB5600208, 1:2,000), β-actin (Sigma-Aldrich, A3854; 1:1000), phosphorylated NF-kB p65 (Novus, NB100-82086, 1:500), phosphorylated STAT1 (R&D Systems, AF2894, 1 μg/ml), phosphorylated STAT3 (Novus, NBP2-24463, 0.5 μg/ml), or phosphorylated STAT6 (Millipore, 06-937, 1:1000).

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Activation Assay, Phospho-proteomics, Western Blot, Software

Journal: Journal of clinical immunology

Article Title: Hematopoietic Stem Cell Transplantation in Patients with Heterozygous STAT1 Gain-of-Function Mutation

doi: 10.1007/s10875-018-0575-y

Figure Lengend Snippet: (A) Pre- and post-transplantation total p-STAT1 expression in CD4+ T cells stimulated with IFN-β (20 ng/mL) by flow cytometry in patient (P1) compared to healthy control. (B) The dose response curve of STAT1 phosphorylation induced with IFN-β in patient and control CD4+ T cells. *** p<0.0001 by two-way ANOVA. Tx: Transplantation.

Article Snippet: Monoclonal antibodies (mAbs) to the following human proteins were used for staining: CD3 (UCHT1), CD4 (RPA-T4), IFN-γ (4S.B3), IL-17 (BL168) (Biolegend), phospho (p)-STAT1 (KIKSI0803), (all from eBioscience), STAT1 (246523; R&D Systems).

Techniques: Transplantation Assay, Expressing, Flow Cytometry, Control, Phospho-proteomics

Journal: Journal of clinical immunology

Article Title: Hematopoietic Stem Cell Transplantation in Patients with Heterozygous STAT1 Gain-of-Function Mutation

doi: 10.1007/s10875-018-0575-y

Figure Lengend Snippet: The details and outcome of HSCT in this study and other studies for STAT1 GOF mutated patients

Article Snippet: Monoclonal antibodies (mAbs) to the following human proteins were used for staining: CD3 (UCHT1), CD4 (RPA-T4), IFN-γ (4S.B3), IL-17 (BL168) (Biolegend), phospho (p)-STAT1 (KIKSI0803), (all from eBioscience), STAT1 (246523; R&D Systems).

Techniques: Mutagenesis, Infection

Journal: Blood Advances

Article Title: Resistance to histone deacetylase inhibitors confers hypersensitivity to oncolytic reovirus therapy

doi: 10.1182/bloodadvances.2020002297

Figure Lengend Snippet: Belinostat-resistant TCL cells exhibit downregulated IRF1 and STAT1 expression. (A) Transcriptome analysis of HuT-78 and Karpas-299 parental and resistant cell lines reveal significant alterations in antiviral genes, including reductions in STAT1 and IRF1. Gene expression changes were determined by using Affymetrix expression arrays. Virus-related genes with significant induction/repression are illustrated in the heat maps. (B-C) Real-time qPCR analysis of STAT1 and IRF1 levels. STAT1 and IRF1 expression was measured by real-time qPCR in parental and belinostat-resistant TCL cells. Data are shown as mean ± SD (n = 3). *P < .05 indicates a significant difference compared with parental cells. (D) STAT1 and IRF1 protein expression is decreased in belinostat-resistant TCL cells. IRF1, phospho-STAT1, and STAT1 protein expression was determined by immunoblotting. (E) Belinostat-resistant cells show reduced IRF1 and STAT1 promoter site binding. ChIP assays were used to quantify the binding of IRF1 and STAT1 to their respective promoters. Absence of Ig (- Ig) served as a negative control, and normal rabbit IgG was used as an isotype control. Histone H3 (H3) served as a positive control, and chromatin input was used as a loading control. NC represents a negative control for the PCR reaction.

Article Snippet: Thus, STAT1 shRNA #1 (Origene) was used for all experiments.

Techniques: Expressing, Gene Expression, Virus, Western Blot, Binding Assay, Negative Control, Control, Positive Control

Journal: Blood Advances

Article Title: Resistance to histone deacetylase inhibitors confers hypersensitivity to oncolytic reovirus therapy

doi: 10.1182/bloodadvances.2020002297

Figure Lengend Snippet: Targeting STAT1 enhances the antilymphoma activity of oncolytic reovirus. (A) Silencing STAT1 expression in TCL cell lines. STAT1 was knocked down in parental Karpas-299 and HuT-78 cells using 3 different lentiviral shRNA constructs, and efficiency was assessed by immunoblotting. STAT1 shRNA #1 was used for all experiments. (B) Reolysin exhibits greater antilymphoma activity in TCL cells with diminished STAT1 levels. TCL cells infected with Control and STAT1 shRNA were treated with the indicated concentrations of Reolysin for 72 hours, and cell viability was determined by MTT assay. Data are shown as mean ± SD (n = 3). *P < .05 indicates a significant difference compared with Control shRNA cells treated with the same concentration of Reolysin. (C) Reovirus viral load is significantly greater in TCL cells with STAT1 knockdown. Karpas-299 and HuT-78 cells were treated for 48 hours with 45 and 90 PFUs Reolysin per cell, respectively. Reovirus accumulation was visualized by electron microscopy. Circles indicate reovirus accumulation. Quantification of reovirus in Control and STAT1 shRNA TCL cells. The number of reovirus particles per cell was quantified using ImageJ software. Data are shown as mean ± SD (n = 5). *P < .05 indicates a significant difference compared with Control shRNA cells treated with Reolysin. (D) Treatment with the JAK inhibitor ruxolitinib sensitizes TCL cells to Reolysin treatment. Parental and belinostat-resistant Karpas-299 and HuT-78 cells were treated with 45 and 90 PFUs Reolysin per cell, respectively, 5 μM ruxolitinib, or the combination for 72 hours, and cell viability was measured by MTT assay. Data are shown as mean ± SD (n = 3). *P < .05 indicates a significant difference compared with Control or **P < .05 for treatment with either ruxolitinib or Reolysin as a single agent.

Article Snippet: Thus, STAT1 shRNA #1 (Origene) was used for all experiments.

Techniques: Activity Assay, Expressing, shRNA, Construct, Western Blot, Infection, Control, MTT Assay, Concentration Assay, Knockdown, Electron Microscopy, Software

Journal: Blood Advances

Article Title: Resistance to histone deacetylase inhibitors confers hypersensitivity to oncolytic reovirus therapy

doi: 10.1182/bloodadvances.2020002297

Figure Lengend Snippet: Belinostat decreases STAT1 and IRF1 expression and significantly enhances the antilymphoma activity of Reolysin. (A) Belinostat treatment decreases STAT1 and IRF1 expression. Parental TCL cells were treated with 5 and 10 μM belinostat for 24 hours. STAT1 and IRF1 expression were determined by immunoblotting. (B-C) Reolysin enhances the activity of belinostat in both parental and belinostat-resistant TCL cells. Cells were treated with 0.25 μM belinostat and 45 or 90 PFUs Reolysin per cell, respectively, for Karpas-299 and HuT-78 and the combination for 72 hours (B) or 48 hours (C). Cell viability was determined by MTT assay and apoptosis was measured by PI-FACS analysis. Data are shown as mean ± SD (n = 3). *P < .05 indicates a significant difference compared with Control or **P < .05 compared with either monotherapy.

Article Snippet: Thus, STAT1 shRNA #1 (Origene) was used for all experiments.

Techniques: Expressing, Activity Assay, Western Blot, MTT Assay, Control

Journal: Blood Advances

Article Title: Resistance to histone deacetylase inhibitors confers hypersensitivity to oncolytic reovirus therapy

doi: 10.1182/bloodadvances.2020002297

Figure Lengend Snippet: The combination of Reolysin and belinostat results in significantly decreased tumor cell proliferation and augmented apoptosis in parental and belinostat-resistant Karpas-299 tumors. (A-B) Immunohistochemical analysis demonstrates that administration of Reolysin and belinostat effectively decreases PCNA-positive cells (A) and induces significant levels of caspase-3–positive cells (B). Percent PCNA and active caspase-3–positive cells were determined by manual counting. Data are shown as mean ± SD (n = 3 random fields). *P < .05 indicates a significant difference from vehicle or **P < .05 for treatment with either belinostat or Reolysin as a single agent. Scale bars represent 50 μm. (C) Belinostat-induced reduction in STAT1 expression increases TCL cell vulnerability to oncolytic reovirus-mediated cell death. The development of belinostat resistance, acute treatment with belinostat, or STAT1 shRNA infection decreases the expression of STAT1. Oncolytic reovirus replicates significantly more effectively in belinostat-resistant cells and when administered in combination with belinostat resulting in enhanced antilymphoma activity.

Article Snippet: Thus, STAT1 shRNA #1 (Origene) was used for all experiments.

Techniques: Immunohistochemical staining, Expressing, shRNA, Infection, Activity Assay