Journal: Frontiers in Microbiology

Article Title: Single-Molecule RNA Sequencing Reveals IFNγ-Induced Differential Expression of Immune Escape Genes in Merkel Cell Polyomavirus–Positive MCC Cell Lines

doi: 10.3389/fmicb.2021.785662

Figure Lengend Snippet: Overview of all differentially expressed genes categorized according to their activity in cancer biology.

Article Snippet: For antibody staining, 1 × 10 6 cells of each MCC cell line were cultured in a six-well plate in the presence or absence of IFNγ (3,000 U/ml) for 72 h. Cells were washed with fluorescence-activated cell sorting (FACS) buffer (dulbecco’s phosphate-buffered saline with 1% FBS and 0.2% sodium azide) and stained for the following markers: STAT1α/β, Indoleamine-2,3-dioxygenase (IDO), C-X-C motif chemokine 10 (CXCL10), PD-L1, bone marrow stromal antigen 2 (BST2), HLA class II histocompatibility antigen gamma chain (CD74), and HLA A, B, and C (HLA-ABC) with the following monoclonal antibodies: anti-human STAT1 (phycoerythine; PE, clone REA272, Miltenyi Biotec), anti-IDO1 (PE, clone eyedio, Invitrogen), anti-human CXCL10 (PE, clone REA334, Miltenyi Biotec), anti-CD274 (Pe-Cy7, clone MIH1, BD Biosciences), anti-human BST2 (PE, clone REA202, Miltenyi Biotec), anti-CD74 (PE, clone 5-329, Miltenyi Biotec), and anti–HLA-ABC (fluorescein-5-isothiocyanat, BD Biosciences).

Techniques: Activity Assay, Ubiquitin Proteomics

Journal: Frontiers in Microbiology

Article Title: Single-Molecule RNA Sequencing Reveals IFNγ-Induced Differential Expression of Immune Escape Genes in Merkel Cell Polyomavirus–Positive MCC Cell Lines

doi: 10.3389/fmicb.2021.785662

Figure Lengend Snippet: Validation of differential gene expression in MCC cell lines on protein level and mRNA level. (A) TPM values were acquired from our sequencing output. The data represent mean values ± SEM from three independent experiments (two for MKL-1 control). (B) After 72 h of treatment with or without IFNγ (3,000 U/ml), the Merkel cell carcinoma cell lines MKL-1, MKL-2, and WaGa were stained for signal transducer and activator of transcription 1-alpha/beta (STAT1), bone marrow stromal antigen 2 (BST2), C-X-C motif chemokine 10 (CXCL10), and HLA class II histocompatibility antigen gamma chain (CD74) and analyzed by flow cytometry. The average MFI of three independent experiments ± standard error is indicated. p -values were determined using the paired student’s t -test. * p < 0.05; ** p < 0.01; ns, not significant.

Article Snippet: For antibody staining, 1 × 10 6 cells of each MCC cell line were cultured in a six-well plate in the presence or absence of IFNγ (3,000 U/ml) for 72 h. Cells were washed with fluorescence-activated cell sorting (FACS) buffer (dulbecco’s phosphate-buffered saline with 1% FBS and 0.2% sodium azide) and stained for the following markers: STAT1α/β, Indoleamine-2,3-dioxygenase (IDO), C-X-C motif chemokine 10 (CXCL10), PD-L1, bone marrow stromal antigen 2 (BST2), HLA class II histocompatibility antigen gamma chain (CD74), and HLA A, B, and C (HLA-ABC) with the following monoclonal antibodies: anti-human STAT1 (phycoerythine; PE, clone REA272, Miltenyi Biotec), anti-IDO1 (PE, clone eyedio, Invitrogen), anti-human CXCL10 (PE, clone REA334, Miltenyi Biotec), anti-CD274 (Pe-Cy7, clone MIH1, BD Biosciences), anti-human BST2 (PE, clone REA202, Miltenyi Biotec), anti-CD74 (PE, clone 5-329, Miltenyi Biotec), and anti–HLA-ABC (fluorescein-5-isothiocyanat, BD Biosciences).

Techniques: Biomarker Discovery, Gene Expression, Sequencing, Control, Staining, Flow Cytometry

Journal: EBioMedicine

Article Title: Mediator Kinase Phosphorylation of STAT1 S727 Promotes Growth of Neoplasms With JAK-STAT Activation

doi: 10.1016/j.ebiom.2017.11.013

Figure Lengend Snippet: Genome-wide mapping of STAT1 pS727 reveals occupancy at super-enhancers. A. GSEA plot shows enrichment of CA-upregulated genes in the top non-promoter STAT1 pS727 regions. B. Venn diagram shows the top STAT1 pS727 peaks overlap significantly with genes upregulated by CA (≥ 1.2-fold), P = 1.05e − 10 and H3K27Ac SEs, P = 1.49e − 60 (hypergeometric tests). C. ChIP-seq tracks at the GATA2 locus comparing normalized STAT pS727, total STAT1, CDK8, and H3K27ac signals (in reads per million) in SET-2 cells. D. Model for the mechanism of growth inhibition by CA in JAK-STAT activated neoplasms. See also Fig. S5.

Article Snippet: Primary antibodies included: STAT1 (CST #9172), STAT1 (R&D Systems #PAF-ST1), STAT1 pS727 (CST #9177), STAT1 pY701 (CST #9167), CDK8 (CST #4101 or #4106), CDK19 (Sigma #HPA007053), CDK9 (CST #2316), FLAG (Sigma F1804), PARP (CST #9532), actin (Sigma #A5060), GAPDH (Santa Cruz sc-47724).

Techniques: Genome Wide, ChIP-sequencing, Inhibition

Journal: EBioMedicine

Article Title: Mediator Kinase Phosphorylation of STAT1 S727 Promotes Growth of Neoplasms With JAK-STAT Activation

doi: 10.1016/j.ebiom.2017.11.013

Figure Lengend Snippet: CA suppresses growth of JAK2-mutant AML by inhibiting phosphorylation of STAT1 S727. A. Immunoblots after 2 h treatment showing dose-dependent inhibition of STAT1 S727 phosphorylation by CA in SET-2 cells. Ruxolitinib (Rux), a JAK1/2 inhibitor, inhibits tyrosine phosphorylation of STAT1 at 1 μm. Combined treatment with 1 μm ruxolitinib and 50 nM CA. Representative blots shown of at least three biological replicates. B. Competitive growth experiments show that SET-2 and UKE-1 cells overexpressing the phosphomimetic mutant STAT1 S727E (ZsGreen) outcompete cells expressing wild-type STAT1 (mCherry) in the presence of CA (mean ± s.e.m. ratio, n = 3 biological replicates). Representative growth curves shown for experiments repeated at least three times. C. A homozygous CRISPR-Cas9-edited clone expressing STAT1 S727E is resistant to CA compared to a control CRISPR knockout line targeting AAVS1. Solid lines represent calculated non-linear fit (STAT1 S727E(GI 50 ) = 37 nM, sgAAVS1(GI 50 ) = 0.3 nM). Dots represent the mean ± s.e.m. ratio of n = 3 biological replicates. D. CA-induced upregulation of CD61 can be inhibited by STAT1 S727E overexpression. CD61 mRNA transcript levels were measured by ddPCR (mean ± Poisson error, n = 5 biological replicates, experiment performed twice, P = 0.000091, two-tailed t -test). E. STAT1 S727A overexpression causes upregulation of CD41 on the cell surface compared to wild-type STAT1 in SET-2 cells. Representative flow cytometry traces shown and quantification plotted on the right (mean ± s.e.m., n = 4 biological replicates). See also Fig. S2.

Article Snippet: Primary antibodies included: STAT1 (CST #9172), STAT1 (R&D Systems #PAF-ST1), STAT1 pS727 (CST #9177), STAT1 pY701 (CST #9167), CDK8 (CST #4101 or #4106), CDK19 (Sigma #HPA007053), CDK9 (CST #2316), FLAG (Sigma F1804), PARP (CST #9532), actin (Sigma #A5060), GAPDH (Santa Cruz sc-47724).

Techniques: Mutagenesis, Phospho-proteomics, Western Blot, Inhibition, Expressing, CRISPR, Control, Knock-Out, Over Expression, Two Tailed Test, Flow Cytometry

Journal: EBioMedicine

Article Title: Mediator Kinase Phosphorylation of STAT1 S727 Promotes Growth of Neoplasms With JAK-STAT Activation

doi: 10.1016/j.ebiom.2017.11.013

Figure Lengend Snippet: Depletion of STAT1α or the STAT1 TAD induces growth arrest in JAK2-mutant AML cells. A. Diagram depicts location of various sgRNAs used to target STAT1. Dotted lines represent locations of the Cas9 cut site for each guide. Inset, part of the TAD (transactivation domain) DNA and protein sequence, with the CDK8 recognition motif highlighted in blue and serine 727 in red. B. CRISPR-Cas9-mediated knockout (KO) of STAT1 using sg1 and sg9n shows efficient depletion of STAT1 by immunoblot. STAT1-KO (mCherry) cells show resistance to CA compared to control AAVS1-KO (AmCyan) cells (mean ± s.e.m. ratio of n = 3 biological replicates). Representative growth curves shown for experiments repeated at least three times. C. The pie charts represent the percentage of frameshit versus in-frame mutations generated in pools of cells edited with guide sg5, sg4, sg11, or sg9n. SET-2 or UKE-1 cells were edited two independent times with each sgRNA and sequenced; one experiment is shown. D. Model for the role of STAT1 in proliferation. A proliferative state is maintained when STAT1α/β are present or absent. Growth arrest is induced when the TAD of STAT1α is inhibited by CA or when STAT1α is knocked out. See also Figs. S3 and S4.

Article Snippet: Primary antibodies included: STAT1 (CST #9172), STAT1 (R&D Systems #PAF-ST1), STAT1 pS727 (CST #9177), STAT1 pY701 (CST #9167), CDK8 (CST #4101 or #4106), CDK19 (Sigma #HPA007053), CDK9 (CST #2316), FLAG (Sigma F1804), PARP (CST #9532), actin (Sigma #A5060), GAPDH (Santa Cruz sc-47724).

Techniques: Mutagenesis, Sequencing, CRISPR, Knock-Out, Western Blot, Control, Generated

Journal: EBioMedicine

Article Title: Mediator Kinase Phosphorylation of STAT1 S727 Promotes Growth of Neoplasms With JAK-STAT Activation

doi: 10.1016/j.ebiom.2017.11.013

Figure Lengend Snippet: CA upregulates the expression of differentiation programs in MPN cells, partly through STAT1. A. Scatterplot of differentially expressed genes in SET-2 cells with CA or ruxolitinib treatment (RNA-seq) shows large differences between the transcriptional effects of the two compounds. Ruxolitinib data from . Best-fit linear regression line in blue, R 2 = 0.000047. B. Graph shows enriched molecular function and biological process terms related to transcription factor activity and differentiation from gene ontology analysis (DAVID) of 261 CA-upregulated genes. C. Table shows positive enrichment of three MK differentiation signatures with genes upregulated by CA treatment, but not ruxolitinib treatment (GSEA). D. GSEA plots show that CA upregulates positively-regulated STAT1 target genes, not negatively-regulated STAT1 target genes (published signatures from ). E. STAT1 S727E overexpression (probed for GATA2 , UBASH3B ) or knockin (probed for rest) partially rescues upregulation of some SE-associated genes in SET-2 cells by ddPCR (mean ± s.e.m. of Poisson error of two independent experiments, n = 6 biological replicates, two-tailed t -tests). See also Fig. S5 and Table S1.

Article Snippet: Primary antibodies included: STAT1 (CST #9172), STAT1 (R&D Systems #PAF-ST1), STAT1 pS727 (CST #9177), STAT1 pY701 (CST #9167), CDK8 (CST #4101 or #4106), CDK19 (Sigma #HPA007053), CDK9 (CST #2316), FLAG (Sigma F1804), PARP (CST #9532), actin (Sigma #A5060), GAPDH (Santa Cruz sc-47724).

Techniques: Expressing, RNA Sequencing, Activity Assay, Over Expression, Knock-In, Two Tailed Test

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 immunology (Baltimore, Md. : 1950)

Article Title: IFN mimetic as a therapeutic for lethal vaccinia virus infection: possible effects on innate and adaptive immune responses.

doi: 10.4049/jimmunol.178.7.4576

Figure Lengend Snippet: FIGURE 7. IFN- mimetic treatment results in activation of STAT1 and nuclear translocation of STAT1 and IFNGR1. a, Nuclear translocation of STAT1 and IFNGR1. WISH cells treated with 10 M lipo-IFN-95–132 (left columns), or lipo-IFN- (95–125) (right columns) were stained simultaneously with Abs to STAT1 and IFNGR1. Secondary Abs to STAT1 conjugated to Alexa 594 (top row), or to IFNGR1 conjugated to Cy-2 (bottom row) were used and analyzed by fluorescence microscopy. b, Phosphorylation of STAT1 by IFN mimetic. Cell extracts from WISH cells, untreated (lane 1), control peptide treated (lane 2), or IFN mimetic treated (lane 3) were electro- phoresed, transferred to Immobilon-P, and probed with an Ab to phospho- STAT1 (top row). The filter was stripped and reprobed with an Ab for total STAT1 (bottom row) to ensure equal loading of protein.

Article Snippet: Slides were then incubated for 1 h in blocking buffer containing rabbit polyclonal antisera against IFNGR1 (Santa Cruz Biotechnology) and goat polyclonal antisera to human STAT1 (R&D Systems).

Techniques: Activation Assay, Translocation Assay, Staining, Microscopy, Phospho-proteomics, Control

Journal: Iranian Journal of Medical Sciences

Article Title: Epstein-Barr Virus Promotes Tumorigenicity and Worsens Hodgkin Lymphoma Prognosis by Activating JAK/STAT and NF-κB Signaling Pathways

doi: 10.30476/IJMS.2023.97287.2896

Figure Lengend Snippet: The sequence of primers used for the real-time polymerase chain reaction

Article Snippet: The expression levels of target genes were then estimated using the 2 −ΔΔCT method., Primers STAT1 (#HP210040), JAK2 (#HP208201), IRF-1 (HP205934), PD-L1 (#HP210654), IFN-γ (#HP200586), NF-κB (#HP207409), Bcl-xL (#HP234144), COX-2 (#HP200900), GAPDH (#HP205798), and β-catenin (#KN208947) were purchased from OriGene Technologies (Beijing, China).

Techniques: Sequencing

Journal: Iranian Journal of Medical Sciences

Article Title: Epstein-Barr Virus Promotes Tumorigenicity and Worsens Hodgkin Lymphoma Prognosis by Activating JAK/STAT and NF-κB Signaling Pathways

doi: 10.30476/IJMS.2023.97287.2896

Figure Lengend Snippet: The mRNA expression of JAK2/STAT1 and NF-κB signaling pathways in patients with EBV-positive and EBV-negative Hodgkin lymphoma

Article Snippet: The expression levels of target genes were then estimated using the 2 −ΔΔCT method., Primers STAT1 (#HP210040), JAK2 (#HP208201), IRF-1 (HP205934), PD-L1 (#HP210654), IFN-γ (#HP200586), NF-κB (#HP207409), Bcl-xL (#HP234144), COX-2 (#HP200900), GAPDH (#HP205798), and β-catenin (#KN208947) were purchased from OriGene Technologies (Beijing, China).

Techniques: Expressing

Journal: Clinical Science (London, England : 1979)

Article Title: Interferon regulatory factor 1 (IRF1) inhibits lung endothelial regeneration following inflammation-induced acute lung injury

doi: 10.1042/CS20220876

Figure Lengend Snippet: ( A ) Representative immunoblots and densitometric quantification of Stat1 Ser727 phosphorylation levels in MLECs isolated from Ifr1 fl/fl control mice pre- and post-LPS. ( B ) Representative immunoblots and densitometric quantification of Stat1 Ser727 phosphorylation levels in MLECs in Ifr1 fl/fl control mice and endothelial cell-specific Irf1 knockout mice at baseline. ( C ) Representative immunoblots and densitometric quantification of Stat1 Ser727 phosphorylation and Irf1 protein levels in the indicated MLEC cell lines following LPS (10 μg/ml) treatment. ( D ) Representative immunoblots and densitometric quantification of STAT1 Ser727 phosphorylation and IRF1 protein levels in the indicated HLMVEC cell lines following LPS (10 μg/ml) treatment. ( E ) Schematic of the human IRF1 promoter region depicting the two highly-conserved STAT1 binding site at −44 ∼ −56 bp and −177 ∼ −188 bp. The WT and MUT GAS1 sequences used in panel ( G ) are provided. ( F ) HLMVECs subjected to vehicle or LPS conditions for 8 hours, followed by ChIP-qPCR assays for detection of STAT1 binding to the two binding sites within the IRF1 promoter region. (G) HLMVECs co-transfected with control or STAT1 plasmid along with one of three luciferase (Luc) reporter gene constructs. Schematic representations of Luc constructs are indicated. All experiments: n = 6 mice or 6 independent biological replicates per group. Data represented as means ± SDs. * P <0.05, ** P <0.01 [(A, C, D) one-way ANOVA with Bonferroni post-hoc tests, and Log-rank Mantel-Cox tests; (B) two-tailed Student’s t -tests; (E, H, I) two-way ANOVA with Bonferroni post-hoc tests].

Article Snippet: For in vitro gene overexpression, the pMXs-ms- Irf1 , pMXs-ms- IRF1 , pMXs-ms- Stat1 , and pMXs-ms- STAT1 plasmids were generated by respectively cloning the mouse Irf1 cDNA clone (NM_008390, cat. no. MC200482, Origene), human IRF1 cDNA clone (NM_002198, cat. no. SC118744, Origene), murine Stat1 cDNA clone (BC004808, cat no. MC200236, Origene), or human STAT1 cDNA clone (NM_007315, cat. no. SC115595, Origene) into the pMXs-GW backbone (plasmid 18656, Addgene).

Techniques: Western Blot, Isolation, Control, Knock-Out, Binding Assay, Transfection, Plasmid Preparation, Luciferase, Construct, Two Tailed Test