Journal: Frontiers in Immunology

Article Title: Transcriptional inhibition of STAT1 functions in the nucleus alleviates Th1 and Th17 cell-mediated inflammatory diseases

doi: 10.3389/fimmu.2022.1054472

Figure Lengend Snippet: Generation of nucleus-deliverable ndSTAT1-TMD and verification of its intra-nuclear transduction kinetics without cellular cytotoxicity. (A) Protein structure of STAT1-TMD without Hph-1-PTD, ndSTAT-TMD, and ndSTAT1-TMD (V426D, T427D) with mutated valine 426, and threonine 427. PTD, protein transduction domain; nd, nucleus-transducible; TMD, transcription modulation domain; DBD, DNA binding domain; ND, N-terminal domain; LD, linker domain; SH2, SCR2 homology domain; TAD, transactivation domain. (B) The identity of the purified STAT1-TMD, ndSTAT1-TMD, and ndSTAT1-TMD (V426D, T427D) was confirmed by western blot (left panel) or SDS-PAGE (right panel). (C, D) Dose-dependent and time-dependent intra-nuclear delivery of ndSTAT1-TMD (0.5-2 μM) or STAT1-TMD (2 μM) into mouse splenocytes. (E) Intracellular stability of ndSTAT1-TMD after transduction into mouse splenocytes. (F) Intracellular localization of ndSTAT1-TMD in cultured mouse splenocytes. Representative immunofluorescence 63X (left panel) and 189X (right panel) images using a confocal microscope. Scale bar = 10 μm. (G) Evaluation of cellular cytotoxicity of ndSTAT1-TMD or ndSTAT1-TMD (V426D, T427D) in mouse splenocytes analyzed by CCK-8 assay. (H, I) The level of the induced expression of CD25, CD69, or IL-2 secretion in CD4 + T cells activated by anti-CD3ϵ antibody and anti-CD28 antibody in the presence of ndSTAT1-TMD or ndSTAT1-TMD (V426D, T427D) was analyzed by flow cytometry and ELISA. The graphs are represented as mean ± SEM (n=3), and the statistical analysis was examined using Student’s t-test. ns, not significant.

Article Snippet: The plasmid of mouse STAT1 (BC004808) was purchased from Origene Technologies, Inc, USA.

Techniques: Transduction, Binding Assay, Purification, Western Blot, SDS Page, Cell Culture, Immunofluorescence, Microscopy, CCK-8 Assay, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Transcriptional inhibition of STAT1 functions in the nucleus alleviates Th1 and Th17 cell-mediated inflammatory diseases

doi: 10.3389/fimmu.2022.1054472

Figure Lengend Snippet: ndSTAT1-TMD inhibits the expression of STAT1-target genes at the transcription level. (A) HEK293T cells were co-transfected with the plasmids containing the luciferase reporter gene, whose expression is driven by the IL-17A promoter, and the wild-type STAT1 gene. The luciferase activity of cells was measured 24 hr after the treatment with ndSTAT1-TMD or ndSTAT1-TMD (V426D, T427D) by a luminometer. Data are represented as mean ± SEM (n=5), and statistical significance was indicated when the luciferase activity was lower than the positive control group (pCMV-STAT1-only). The statistical analysis was examined using Student’s t-test. ****p<0.0001. (B, C) GSEA-based enrichment plots of the hallmark of IFN-γ response gene set in ndSTAT1-TMD-treated Th1 cells or of the gene set upregulated in STAT3 knockout CD4 + T cells compared to wild-type CD4 + T cells in ndSTAT1-TMD-treated Th17 cells. (D, E) Heatmap of Th1 or Th17 cell signature genes and naïve T cell-specific genes in Th1 or Th17 cells treated with or without ndSTAT1-TMD. Each column means results from one experimental well Z-score was calculated from log2(TPM+1).

Article Snippet: The plasmid of mouse STAT1 (BC004808) was purchased from Origene Technologies, Inc, USA.

Techniques: Expressing, Transfection, Luciferase, Activity Assay, Positive Control, Knock-Out

Journal: Theranostics

Article Title: H3K27 acetylation activated-COL6A1 promotes osteosarcoma lung metastasis by repressing STAT1 and activating pulmonary cancer-associated fibroblasts

doi: 10.7150/thno.51245

Figure Lengend Snippet: COL6A1 promotes OS invasion and migration via suppressing STAT1 expression and activation. A. Scatter plot of top 20 KEGG pathways enrichment of DEGs after COL6A1 transfection. Rich factor is the ratio of the DEG number to the background number in a certain pathway. The size of the dots represents the number of genes, and the color of the dots represents the range of the q-value. B. STAT1 and STAT3 mRNA levels were determined in OS cell with COL6A1 or siCOL6A1 transfection by qRT-PCR. C. Total and phosphorylated STAT1 and STAT3 protein expression levels were analyzed in COL6A1 overexpression and knockdown in OS cells by western blot. D. Nuclear and cytoplasmic STAT1 expression was analyzed in COL6A1 overexpression or control OS cells. E. STAT1 transcription luciferase reporter constructs were transiently transfected into the indicated cells, and luciferase activity was analyzed after 48 hours. F. Biotin pull-down assay with a STAT1 probe was used to determine its DNA binding after transfecting COL6A1. G. STAT1 overexpression decreased the migratory ability of COL6A1 overexpression OS cells. The migratory ability of OS cells was detected upon the indicated treatment (right panel). H . The expression of STAT1 in OS tissues was detected by western blot. I. Expression of STAT1 and COL6A1 in OS tissues was detected by immunohistochemistry and COL6A1 inversely correlated with STAT1 expression in human OS tissues (Scale bars: 100 µm). J. Overexpression of STAT1 decreased the rate of lung metastasis after tail-vein injection indicated cells (n = 6 each group). Representative photographs of H&E staining in lung metastases tissues from mice orthotopically inoculated with indicated treated cells (Scale bars: 400 µm). Data represent the mean ± SD of 3 separate determinations. * p < 0.05, ** p < 0.01, *** p < 0.001 by Student's t test.

Article Snippet: GFP-tagged wild type STAT1 (wt), GFP-tagged STAT1 (Y701A), and GFP-tagged STAT1 (S727A) were purchased from Addgene (Watertown, MA, USA).

Techniques: Migration, Expressing, Activation Assay, Transfection, Quantitative RT-PCR, Over Expression, Western Blot, Luciferase, Construct, Activity Assay, Pull Down Assay, Binding Assay, Immunohistochemistry, Injection, Staining

Journal: Theranostics

Article Title: H3K27 acetylation activated-COL6A1 promotes osteosarcoma lung metastasis by repressing STAT1 and activating pulmonary cancer-associated fibroblasts

doi: 10.7150/thno.51245

Figure Lengend Snippet: COL6A1 interacted with E3 ligase SOCS5 to promote STAT1 degradation. A. Co-localization of COL6A1 and STAT1 in ESCC cells were examined by using confocal microscopy (scale bar, 10 µm). B. The interaction of GFP-STAT1 and Flag-COL6A1 wide type, VWFA1, VWFA2, VWFA3 domains deletion mutations were detected by co-immunoprecipitation in 293T cells. C. COL6A1 was transfected into the OS cells in the presence of cycloheximide (CHX, 200 µg/mL) for indicated times. Cell lysates were immunoblotted by antibodies as indicated. The data were quantified using Image J software. D. STAT1 ubiquitination was detected by immunoprecipitation with anti-STAT1 antibody and immunoblotting with an anti-Ub antibody. E. The interaction of STAT1, COL6A1 and SOCS5 was detected by co-immunoprecipitation in U2OS. F. SOCS5 decreased STAT1 protein. U2OS cells were transfected with Flag-SOCS5 or Flag-SOCS5 R406K as well as control or COL6A1 transfection. The protein expression level of STAT1 was assayed by western blot. G . The cells expressing wide type SOCS5 were treated with CHX. The protein levels of STAT1 and SOCS5 were analyzed by western blot. H . Knockdown SOCS5 increased STAT1 protein. U2OS cells were transfected with si-SOCS5 as well as control or COL6A1 transfection. I . U2OS cells were transfected with control or SOCS5 siRNAs treated with CHX (200 µg/mL), the protein levels of STAT1 and SOCS5 were analyzed by western blot. J. SOCS5 ubiquitylates STAT1. U2OS cells were transfected with indicated plasmids or siRNA for 48 h. Cell lysates were immunoprecipitated with anti-GFP and analyzed by immunoblotting with indicated antibodies. K . U2OS cells were transfected with indicated plasmids. lysates were immunoprecipitated with anti-GFP, and western blots were performed to analyze the presence of indicated proteins and levels of ubiquitination. Data represent the mean ± SD of 3 separate determinations. * p < 0.05, ** p < 0.01, *** p < 0.001 by Student's t test.

Article Snippet: GFP-tagged wild type STAT1 (wt), GFP-tagged STAT1 (Y701A), and GFP-tagged STAT1 (S727A) were purchased from Addgene (Watertown, MA, USA).

Techniques: Confocal Microscopy, Immunoprecipitation, Transfection, Software, Western Blot, Expressing

Journal: Theranostics

Article Title: H3K27 acetylation activated-COL6A1 promotes osteosarcoma lung metastasis by repressing STAT1 and activating pulmonary cancer-associated fibroblasts

doi: 10.7150/thno.51245

Figure Lengend Snippet: Activated fibroblasts promote OS metastasis by secreting TGF-βA. The cell invasion ability was detected by transwell assay after TGF-β treatment. B. COL6A1 expression was detected by western blot, confocal microscope and qRT-PCR in OS cells exposed to mock vehicle or TGF-β (0-10 ng/mL) for 12 h. C . COL6A1 protein and mRNA expression in U2OS cells transfected with siRNAs targeting SMAD2 exposed to mock vehicle or TGF-β (5 ng/mL) for 12 h. D. COL6A1-knockdown Saos-2 cells were treated with TGF-β, western blot were performed to detect the expression of COL6A1. E . COL6A1-knockdown Saos-2 cells were treated with TGF-β and transwell assay were performed to detect the migration ability of OS cells. F. The expressions of E-cadherin, N-cadherin, β-catenin and vimentin in OS cells with COL6A1 or si-COL6A1 transfection by western blot, qRT-PCR and confocal microscope (Scale bars: 50 µm). H. COL6A1-knockdown U2OS cells were treated with TGF-β, western blots and qRT-PCR were performed on cell lysates with the indicated antibodies . The protein and mRNA expression of TGF-β was detected in COL6A1 overexpressed OS cells. I. The concentration of TGF-β in OS patients' serum was detected by ELISA (non-Meta: non-metastasis OS patients; Meta: lung metastasis OS patients; H.C.: healthy control). J. Schematic diagram summarizing how COL6A1 promotes OS metastasis via suppressing STAT1 expression and activating CAFs. Exo, exosome. Data represent the mean ± SD of 3 separate determinations. * p < 0.05, ** p < 0.01, *** p < 0.001 by Student's t test.

Article Snippet: GFP-tagged wild type STAT1 (wt), GFP-tagged STAT1 (Y701A), and GFP-tagged STAT1 (S727A) were purchased from Addgene (Watertown, MA, USA).

Techniques: Transwell Assay, Expressing, Western Blot, Microscopy, Quantitative RT-PCR, Transfection, Migration, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Dietary Lauric Acid Suppresses Inflammation, Cholestasis, Hepatocyte Injury, and Senescence in 3,5-Diethoxycarbonyl-1,4-Dihydrocollidine-induced Inflammatory Cholangiopathy

doi: 10.1016/j.jcmgh.2026.101731

Figure Lengend Snippet: Effect of lauric acid on expression of inflammation, senescence, and oxidative stress genes and biomarkers in hepatocytes isolated from experimental mouse groups. qRT-PCR-mRNA analysis in hepatocytes isolated from chow, DDC, LA, and DDC/LA mice. ( A ) Genes involved in inflammation and oxidative stress. ( B ) Senescence genes. ( C ) Mitochondrial fatty acid oxidation and oxidative stress genes. ( D ) β-gal staining ( yellow arrow ) as a senescence marker in overnight cultured primary hepatocytes isolated from chow, DDC, LA, and DDC/ LA mice. Bar graph showing the number of senescent (β-gal + ) cells per microscopic field in liver sections from chow, DDC, DDC/LA, and LA groups. ( E ) Immunoblotting and quantification of β-gal in fresh hepatocytes isolated from chow, DDC, LA, and DDC/LA mice. ( F ) Serum hydrogen peroxide levels and CCL2 concentrations (ELISA), and lipid peroxides, nitrite, and malondialdehyde levels from isolated hepatocytes. ( G–H ) Co-culture of human cholangiocytes H69 ( upper wells ) and Huh7 cells was conducted. H69 cells were incubated with and without DDC overnight in the presence or absence of DLPC. ( G ) mRNA expression by qPCR was measured in Huh7 cells ( bottom wells ) for CCL2, CDKN1A, NR0B2, and ABCB11 and ( H ) in H69 cells for CCL2, CDKN1A, KRT19, CTGF, and TNF . ( I ) Primary mouse hepatocytes were incubated with CCL2 in the presence or absence of DLPC overnight, and mRNA expression by qPCR was analyzed for senescence genes Cdkn1a and Cdkn1b . ( J ) β-gal staining ( yellow arrow = senescent cells) of cultured Huh7 cells incubated with CCL2 overnight in the presence or absence of DLPC. Bar graph showing the number of senescent (β-gal + ) Huh7 cells after treatment with CCL2 alone or in combination with DLPC. CCL2 treatment markedly increased cellular senescence, which was inhibited by DLPC. ( K ) Confocal fluorescence microscopy of primary cultured hepatocytes from chow, DDC, LA, and DDC/LA mice stained with senescence marker P16 ( red ), and nuclear DAPI ( blue ) from showing increased senescence in DDC mouse hepatocytes, which was prevented by LA treatment. ( L ) Confocal fluorescence microscopy of primary mouse hepatocytes from chow, DDC, LA, and DDC/LA mice stained with senescence marker P21/WAF/CIP ( red ) and nuclear DAPI ( blue ) also showing increased senescence in DDC mouse hepatocytes, which was prevented by LA treatment. For ( G–I ), data points represent replicates in 3 independent experiments. For ( D and J ), photomicrographs are shown that are representative of 3 separate experiments. For ( K and L ), immunofluorescent images are shown that are representative of 3 separate experiments. ( M ) Western analysis of pSTAT1 protein, total STAT1, and actin expression in hepatocytes isolated from the mouse groups, including quantification of integrated density values (IDVs). ( N ) ChIP assay of hepatocytes isolated from mouse groups for STAT1 binding to the promoter region of Cdkn1b using a STAT1-specific antibody. ( O ) Western analysis of pSTAT1 protein expression in cultured primary mouse hepatocytes exposed to CCL2 overnight in the presence or absence of DLPC, including quantification of IDVs of immunoblots. Statistical analysis was performed by 1-way ANOVA with Tukey’s correction for multiple comparisons. a P < .05 vs all other groups; b P < .05 vs DDC; b P < .05 vs CCL2.

Article Snippet: Phospho-STAT1 , 9167 , 1:1000 , Cell Signaling.

Techniques: Expressing, Isolation, Quantitative RT-PCR, Staining, Marker, Cell Culture, Western Blot, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Incubation, Fluorescence, Microscopy, Binding Assay

Journal: eLife

Article Title: Tuberculosis-associated IFN-I induces Siglec-1 on tunneling nanotubes and favors HIV-1 spread in macrophages

doi: 10.7554/eLife.52535

Figure Lengend Snippet:

Article Snippet: Antibody , Rabbit monoclonal anti‐human STAT1 (clone 42H3) , Cell Signaling Technology , Cat# 9175 RRID: AB_2197984 , WB (1:100).

Techniques: Derivative Assay, Cell Culture, Transfection, Construct, Enzyme-linked Immunosorbent Assay, Blocking Assay, Control, Recombinant, Isolation, Western Blot, Fluorescence, Software, Imaging

Journal: Bioorganic chemistry

Article Title: Bromo-substituted indirubins for inhibition of protein kinase-mediated signalling involved in inflammatory mediator release in human monocytes.

doi: 10.1016/j.bioorg.2024.107470

Figure Lengend Snippet: Fig. 3. Impact of indirubins 1 and 2 on the activation status of protein kinase pathways. A-F) Human monocytes were pre-treated with indirubins 1 or 2 at the indicated concentrations, or with inhibitors of GSK-3β (SB216763, 5 µM), CDK8 (CCT-251921, 40 nM), CDK9 (JSH-150, 20 nM) or CDK1 (Ro-3306, 10 µM) for 15 min prior to stimulation with LPS 100 ng/mL for 18 h. (A) Protein expression of β-catenin compared to GAPDH as housekeeping protein. (B) Levels of phosphorylated GSK-3β compared to total expression of GSK-3β protein. (C,D) Levels of phosphorylated STAT1 (C, Ser727 and D, Tyr701) vs. β-actin. (E) Levels of phosphorylated NF-κB (Ser276) compared to GAPDH. (F) Levels of phosphorylated Rb (Ser807/Ser811) vs. β-actin. Protein amounts were evaluated by Western Blot and densito metric analysis thereof. n = 3–4 biological replicates. Statistics: Data are shown as A-E) individual values and means ± SEM. Statistics were calculated by one-way ANOVA for multiple comparisons with Dunnett’s correction, testing treatments against vehicle (0.1 % (v/v) DMSO). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant.

Article Snippet: Membranes were incubated with the following primary antibodies: rabbit monoclonal anti-COX-2, 1:1000 (12282, Cell Signaling, Danvers, MA), mouse monoclonal anti-β-catenin, 1:1000 (610153, BD Biosciences, San Jose, CA) mouse monoclonal anti-GSK-3β, 1:1000 (9832, Cell Signaling), rabbit monoclonal anti-phospho-GSK-3β (Ser9), 1:1000 (9323S, Cell Signaling), rabbit monoclonal anti-phosphoSTAT1 (Ser727), 1:1000 (ab109461, Abcam, Cambridge, UK), rabbit monoclonal anti-phospho-STAT1 (Tyr701), 1:1000 (ab109457, Abcam), rabbit monoclonal anti-phospho-NF-κB p65 (Ser276), 1:1000 (ab183559, Abcam), rabbit monoclonal anti-phospho-Rb (Ser807/811), 1:1000 (9308S, Cell Signaling), monoclonal anti-β-actin, 1:1000 (4970, Cell Signaling), mouse monoclonal anti-GAPDH, 1:1000 (97166S, Cell Signaling).

Techniques: Activation Assay, Expressing, Western Blot

Journal: Cell reports

Article Title: Tumor-specific CD4 T cells instruct monocyte fate in pancreatic ductal adenocarcinoma

doi: 10.1016/j.celrep.2023.112732

Figure Lengend Snippet: (A) NicheNet analysis from scRNA-seq data from using CD4 T cells as donor cells and MHCII hi macrophages as acceptor cells. See also . (B) Stat1 staining and MFI in MHCII hi and MHCII lo intratumoral macrophages at day 7. Data are mean ± SEM; n = 4 mice per group; ****p < 0.0001; Student’s t test. (C) Tumor weights from WT or Ifngr1 −/− mice; n = 4–8 mice per group; ****p < 0.0001; Student’s t test for each time point. (D) Proportion of MHCII hi or MHCII lo macrophages from WT or Ifngr1 −/− mice. Data are mean ± SEM; n = 4–8 mice per group; ****p < 0.0001; Student’s t test for each time point. (E) Proportion of Arg1 + macrophages on day 7. Data are mean ± SEM; ****p < 0.0001; Student’s t test. (F) Proportion of MHCII lo CD206 + FRβ + macrophages from WT or Ifngr1 −/− mice. Data are mean ± SEM; n = 4–8 mice per group; ****p < 0.0001; Student’s t test for each time point. (G) PD-L1 MFI gated on total macrophages from WT or Ifngr1 −/− mice. Data are mean ± SEM; **p < 0.005; Student’s t test for each time point. (H) Normalized expression of selected genes from bulk RNA sequencing from sorted live MHCII hi or MHCII lo tumor macrophages from WT and Ifngr1 −/− mice at day 14. See also . (I) Gene set enrichment analysis (GSEA) of the top 300 differentially expressed genes from sorted cells in (H). (J) Orthotopic KPC 2a tumor weights at day 7 from WT control mice, WT mice treated with CD40L blockade (αCD40L), or Cd40 −/− mice. *p < 0.05; one way ANOVA with Tukey’s post test. See also . (K) Proportion of MHCII hi or MHCII lo macrophages. Each dot is an independent mouse. Data are mean ± SEM; n = 4–5 mice per group; ***p < 0.001; one-way ANOVA with Tukey’s posttest. (L) Proportion of Arg1 + macrophages. Each dot is an independent mouse. Data are mean ± SEM; n = 4–5 mice per group; ***p < 0.001; one-way ANOVA with Tukey’s posttest. (M) Proportion of MHCII lo CD206 + FRβ + macrophages. Each dot is an independent mouse. Data are mean ± SEM; n = 4–5 mice per group; ***p < 0.001; one-way ANOVA with Tukey’s posttest. See also .

Article Snippet: anti-mouse Stat1 (clone D1K9Y) , Cell Signaling , Cat#: 80916S.

Techniques: Staining, Expressing, RNA Sequencing, Control

Journal: Cell reports

Article Title: Tumor-specific CD4 T cells instruct monocyte fate in pancreatic ductal adenocarcinoma

doi: 10.1016/j.celrep.2023.112732

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: anti-mouse Stat1 (clone D1K9Y) , Cell Signaling , Cat#: 80916S.

Techniques: Recombinant, Red Blood Cell Lysis, Cell Isolation, Cell Stimulation, Software