Journal: Cell Death & Disease

Article Title: Hippo kinases Mst1 and Mst2 maintain NK cell homeostasis by orchestrating metabolic state and transcriptional activity

doi: 10.1038/s41419-024-06828-x

Figure Lengend Snippet: A , B Flow cytometry analysis (left) and MFI intensity (right) demonstrate the expression levels of p-STAT5, p-S6, p-AKT473 and p-Erk in NK cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice upon stimulation with or without IL-15 (50 ng/mL) for 30 min ( n ≥ 4). C Gene-set enrichment analysis of RNA-seq data shows the enrichment of hallmark-IL6-JAK-STAT3-signaling related genes in Mst1/2-deficient NK cells compared to WT NK cells. D Flow cytometry analysis (left) and MFI intensity (right) reveals the expression level of p-STAT3 (Ser727) in NK cells from Mst1/2 fl/fl and Mst1/2 fl/fl CD122 Cre mice upon stimulation with or without IL-15 (50 ng/mL) for 30 min ( n = 4). E ChIP-qPCR analysis demonstrated binding of p-STAT3 conserved motifs in the Tcf7 5′ regulatory region (−17.5 kb), while no binding was observed in a region without p-STAT3-binding motifs (+0.2 kb), serving as a negative control. The experiments were using sorted NK cells from WT mice with anti-p-STAT3 antibody or isotype-matched IgG. F – I WT splenocytes were treated with DMSO, Mst1 inhibitor XMU-MP-1 (1 mM, MedChemExpress) or p-STAT3 inhibitor Stattic (1 mM, MedChemExpress) for 24 h, followed by flow cytometry analysis. The representative plots (left) and summary data (right) show the expression level of p-STAT3 (F), TCF1 ( G ), total cellular ROS ( H ) and the percentage of 7AAD + NK cells ( I ) ( n = 4). J Schematic illustration of Mst1/2 regulate NK cell survival and function via controlling metabolic state and transcriptional activity. Data A , B and D – I are representative of three independent experiments with similar results.

Article Snippet: F – I WT splenocytes were treated with DMSO, Mst1 inhibitor XMU-MP-1 (1 mM, MedChemExpress) or p-STAT3 inhibitor Stattic (1 mM, MedChemExpress) for 24 h, followed by flow cytometry analysis.

Techniques: Flow Cytometry, Expressing, RNA Sequencing, ChIP-qPCR, Binding Assay, Negative Control, Activity Assay

Journal: Virology Journal

Article Title: Influenza a virus regulates interferon signaling and its associated genes; MxA and STAT3 by cellular miR-141 to ensure viral replication

doi: 10.1186/s12985-023-02146-4

Figure Lengend Snippet: Oligonucleotides sequences used for quantifying miR-141 and mRNA of indicated genes

Article Snippet: For staining MxA and STAT3, the primary antibodies; rabbit polyclonal anti-MxA (Novus Biologicals, NBP132905) and mouse monoclonal anti-STAT3 (Abcam, ab119352) were used.

Techniques:

Journal: Virology Journal

Article Title: Influenza a virus regulates interferon signaling and its associated genes; MxA and STAT3 by cellular miR-141 to ensure viral replication

doi: 10.1186/s12985-023-02146-4

Figure Lengend Snippet: The correlation between miR-141 level and the expression profile of MxA and STAT3 in infected A549 cells. (A) Quantification of steady-state miR-141 in infected A549 cells with MOI of 0.5 and transfected with either pre-miR-141 or miR-141 inhibitor compared with noninfected cells (control) using qRT-PCR. (B) Relative gene expression of MxA and STAT3 in infected A549 cells transfected with either specific inhibitor against miR-141 or pre- miR-141 compared with control-transfected cells using qRT-PCR. Error bars indicate the STD of three independent experiments. Student two-tailed t -test used for statistical analysis, (*) indicates P-values ≤ 0.05, and (**) indicates P ≤ 0.01. (C) Flow cytometric assay quantifies the kinetic proteins expression profile of MxA (in blue dots) and STAT3 (in red dots) in infected and transfected A549 cells compared with control cells. (D) Western blot analysis reveals the protein expression level of MxA and STAT3 in infected and transfected cells compared to control cells, β-actin expression profile severed as an internal control

Article Snippet: For staining MxA and STAT3, the primary antibodies; rabbit polyclonal anti-MxA (Novus Biologicals, NBP132905) and mouse monoclonal anti-STAT3 (Abcam, ab119352) were used.

Techniques: Expressing, Infection, Transfection, Control, Quantitative RT-PCR, Gene Expression, Two Tailed Test, Flow Cytometry, Western Blot

Journal: Virology Journal

Article Title: Influenza a virus regulates interferon signaling and its associated genes; MxA and STAT3 by cellular miR-141 to ensure viral replication

doi: 10.1186/s12985-023-02146-4

Figure Lengend Snippet: Quantification analysis of miR-141, MxA, and STAT3 in transfected and infected A549 cells

Article Snippet: For staining MxA and STAT3, the primary antibodies; rabbit polyclonal anti-MxA (Novus Biologicals, NBP132905) and mouse monoclonal anti-STAT3 (Abcam, ab119352) were used.

Techniques: Transfection, Infection, Expressing, Control

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: BMC Medicine

Article Title: ISG15-driven immune modulation and tumor progression in breast cancer metastasis: insights from single-cell and spatial transcriptomics

doi: 10.1186/s12916-025-04614-w

Figure Lengend Snippet: Transcriptomic analysis of in situ tumor tissues in mouse mammary gland cancer. Note: A The volcano plot of differentially expressed genes in in situ tumor tissues of mouse mammary gland cancer after ISG15 silencing, with red dots representing upregulated genes and blue dots representing downregulated genes; B heatmap depicting the expression levels of differentially expressed genes in the sh-NC group (6 cases) and sh-ISG15 group (6 cases), with the left dendrogram clustering genes based on expression levels, the color bar on the right representing expression levels (red indicating upregulated genes and blue indicating downregulated genes), and the histogram above indicating blue for non-metastatic group and red for metastatic group; C GO analysis of differentially expressed genes, with the color bar on the right representing enrichment scores for different functional categories; D KEGG analysis of differentially expressed genes, with the color bar on the right representing enrichment scores for different pathways; E expression levels of PD-L1 in in situ tumor tissues of mouse mammary gland cancer after ISG15 silencing, with 6 mice in each group; F binding sites of transcriptional activation factor STAT3

Article Snippet: The membrane was blocked with 5% skim milk at room temperature (RT) for 1 h, then incubated overnight at 4 °C with primary antibodies against ISG15 (1:1000, ab308219), NESTIN (1:1000, ab221660), OCT4 (1:10,000, ab200834), SOX2 (1:1500, ab92494), STAT3 (1:1500, ab68153), p-JAK2 (1:1500, ab32101), JAK2 (1:5000, ab108596), PD-L1 (1:1000, ab213480), Arg (1:1000, ab203490), CD206 (1:1200, ab64693), CD163 (1:1000, ab182422), CD86 (1:1200, ab220188), and GAPDH (1:2500, ab9485) as the internal reference (all from Abcam, Cambridge, UK), as well as p-STAT3 (1:1000, NB100-82213, Novus Biologicals).

Techniques: In Situ, Expressing, Functional Assay, Binding Assay, Activation Assay

Journal: BMC Medicine

Article Title: ISG15-driven immune modulation and tumor progression in breast cancer metastasis: insights from single-cell and spatial transcriptomics

doi: 10.1186/s12916-025-04614-w

Figure Lengend Snippet: Influence of ISG15 in CSCs on T cell activation and its potential molecular mechanisms. Note: A Flow cytometry analysis of Ki-67 to determine the proportion of proliferating CD8 + T cells; B flow cytometry analysis of IFN-γ to determine the proportion of activated CD8 + T cells; C western blot analysis of JAK-STAT signaling pathway-related proteins and PD-L1 expression in co-culture systems of different groups; D dual-luciferase assay to examine the impact of ISG15 on PD-L1 promoter activity; E the performance of ChIP assay to examine the enrichment of p-STAT3 on the PD-L1 promoter in 4T1-S cells (left panel) and CSCs isolated from mouse tumor models (right panel); F the utilization of flow cytometry for the detection of Ki-67 expression in T cells; G The application of flow cytometry for the detection of IFN-γ expression in T cells; H the use of flow cytometry for the detection of Ki-67 expression in T cells; I the use of flow cytometry for the detection of IFN-γ expression in T cells; J the performance of western blot for the analysis of PD-L1 expression in each group co-culture system; K schematic representation of the molecular mechanism of ISG15 in T cell activation in CSCs. * indicates statistical significance ( P < 0.05) between the two groups. All cellular experiments were performed in triplicate

Article Snippet: The membrane was blocked with 5% skim milk at room temperature (RT) for 1 h, then incubated overnight at 4 °C with primary antibodies against ISG15 (1:1000, ab308219), NESTIN (1:1000, ab221660), OCT4 (1:10,000, ab200834), SOX2 (1:1500, ab92494), STAT3 (1:1500, ab68153), p-JAK2 (1:1500, ab32101), JAK2 (1:5000, ab108596), PD-L1 (1:1000, ab213480), Arg (1:1000, ab203490), CD206 (1:1200, ab64693), CD163 (1:1000, ab182422), CD86 (1:1200, ab220188), and GAPDH (1:2500, ab9485) as the internal reference (all from Abcam, Cambridge, UK), as well as p-STAT3 (1:1000, NB100-82213, Novus Biologicals).

Techniques: Activation Assay, Flow Cytometry, Western Blot, Expressing, Co-Culture Assay, Luciferase, Activity Assay, Isolation