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: 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: Journal for immunotherapy of cancer

Article Title: Orlistat facilitates immunotherapy via AKT-FOXO3a-FOXM1-mediated PD-L1 suppression.

doi: 10.1136/jitc-2024-008923

Figure Lengend Snippet: Figure 3 Orlistat suppressed PD-L1 via inhibition of FOXM1 signaling. (A) Volcano plot of the expression of the reported transcription factors that regulated the expression of PD-L1. These TFs include STAT1, ATF3, IRF2, TFEB, IRF1, YY1, STAT3, JUN, HSF1, NFκB, SP1, FOXP3, BRD4, EZH2, MYC, HIF-1α, and FOXM1. (B, C) qRT-PCR measurement of mRNA expression of FOXM1 after orlistat (40 µM) treatment for 0 hour, 24 hours, or 48 hours in AGS and HCT116 (B), MC38 and CT26 cells (C). (D) WB analysis of FOXM1 protein levels after orlistat treatment for different times (0 hour, 24 hours, 48 hours) in AGS, HCT116, MC38, and CT26 cells. (E) Representative images of immunohistochemical staining of FOXM1 protein expression in resected tumors at day 25. Scale bar=50 µm. (F) The expression of PD-L1 mRNA in AGS and MC38 cells after transfection with FOXM1 overexpression or knockdown. (G) WB for PD-L1 protein levels after FOXM1 overexpression or knockdown in AGS and MC38 cells. (H) Schematic representation of the PD-L1 gene promoter regions and primer pairs used for ChIP assays. (I) ChIP-qPCR assay was used to detect the binding of FOXM1 to the PD-L1 promoter at the (+222 to +450 bp) region in AGS cells. (J) ChIP-qPCR analysis of the impact of orlistat (40 µM) on the binding of FOXM1 to the PD-L1 promoter. (K) The luciferase activity of PD-L1 promoter after FOXM1 overexpression with or without orlistat (40 µM). HEK293T cells in 24-well plates were transfected with FOXM1 plasmids or control. The luciferase activity was measured 36 hours later. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Following activation, STAT1 (TargetMol, TMPJ- 00961) was immobilized on the sensor chip surface using a sodium acetate buffer (pH 5.0).

Techniques: Inhibition, Expressing, Quantitative RT-PCR, Immunohistochemical staining, Staining, Transfection, Over Expression, Knockdown, ChIP-qPCR, Binding Assay, Luciferase, Activity Assay, Control

Journal: Journal for immunotherapy of cancer

Article Title: Orlistat facilitates immunotherapy via AKT-FOXO3a-FOXM1-mediated PD-L1 suppression.

doi: 10.1136/jitc-2024-008923

Figure Lengend Snippet: Figure 8 Orlistat upregulates ISGs and MHC-I through activating the STAT1 pathway. (A) Gene set enrichment plots of the antigen processing and presentation hallmarks. (B) Gene set enrichment plots of the JAK-STAT signaling pathway hallmarks. (C–E) WB analysis of Y701-phosphorylated STAT1 (p-STAT1-701) and total STAT1 in AGS and MC38 cells at different times (C) and concentrations (D) after orlistat administration, and tumors collected from mice-bearing subcutaneous MC38 tumors with or without orlistat treatment (E). (F–K) Quantitative PCR analysis of mRNA expression of IFNα, IFNβ, and ISGs or antigen processing and presentation genes in purified MC38 syngeneic tumors cells treated with control or orlistat (F), MC38 (H, I), and AGS (J, K) cell lines. (L) IB analysis and quantitative data of STAT1 in DMSO control vs orlistat-treated cells. AGS cells were treated with cycloheximide (CHX) 100 mg/mL, and collected at the indicated times. (M) Orlistat administration (40 µM) increased the thermal stability of STAT1 protein as determined by thermal stability shift assay (n=3). (N, O) SPR sensorgrams (N) and steady-state curves (O) for the orlistat analyte (2-fold dilutions; 50–1.56 µM) binding to the STAT1 protein ligand. (P) Schematic diagram depicting orlistat-mediated inhibition of PD-L1 expression in gastric cancer and colon cancer cells and promoting the expression of ISGs and MHC-I **p<0.01, ***p<0.001.

Article Snippet: Following activation, STAT1 (TargetMol, TMPJ- 00961) was immobilized on the sensor chip surface using a sodium acetate buffer (pH 5.0).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Control, Shift Assay, Binding Assay, Inhibition

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