phospho stat1 tyr701  (Cell Signaling Technology Inc)

Journal: Journal of Translational Autoimmunity

Article Title: Exploring the immunomodulatory effects of environmental contaminants on autoimmune patients: An in vitro approach

doi: 10.1016/j.jtauto.2025.100341

Figure Lengend Snippet: Modulation of intracellular signaling pathways in HD, RA, and SLE cells exposed to environmental contaminants. Heatmap showing the relative expression and phosphorylation levels of key signaling proteins involved in immune regulation, inflammation, and cell survival (STAT1, STAT3, p38, AKT, and NFκB) and their phosphorylated forms in HD, RA, and SLE after exposure to PM, silica, and TCDD. Data are represented as z-score normalized values. Asterisks indicate statistically significant differences compared with the unstimulated control within each group (p < 0.05). AKT: protein kinase B; HD: healthy donors; NFκB: nuclear factor kappa-light-chain-enhancer of activated B cells; P: phosphorylated form; p38: p38 mitogen-activated protein kinase; PM: particulate matter; RA: rheumatoid arthritis; SLE: systemic lupus erythematosus; STAT: signal transducer and activator of transcription; TCDD: 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Article Snippet: Membranes were blocked with 5 % non-fat milk in TBS-T and incubated overnight at 4 °C with primary antibodies against phospho-AKT (Thr308), phospho-NFκB p65 (Ser536), phospho-p38 MAPK (Thr180/Tyr182), phospho-STAT1 (Tyr701), and phospho-STAT3 (Tyr705) (Cell Signaling Technology, Danvers, MA, USA; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Protein-Protein interactions, Expressing, Phospho-proteomics, Control

Journal: Redox Biology

Article Title: Hyodeoxycholic acid attenuates atherosclerosis by antagonizing FXR and modulating the PD-1/mTORC1 signaling axis

doi: 10.1016/j.redox.2026.104096

Figure Lengend Snippet: HDCA-treated ameliorate inflammation and modulate lipid metabolism in vivo. (A) Acetyl-CoA levels decreased over time in HDCA-treated Tregs. (B – E) Quantitative real-time PCR analysis of the mRNA expression of key lipogenic genes, including (B) fatty acid synthase (FASN), (C) acetyl-CoA carboxylase (ACC), (D) SCD1, and (E) SREBP-1c, in Tregs with or without HDCA treatment. (F) Functional metabolic flux assay using 13 C-glucose tracing shows decreased enrichment of labeled β-hydroxypalmitate in the HDCA-treated group. (G) Immunoblot analysis of phosphorylated STAT1 in Tregs following HDCA exposure, and quantification statistical analysis results are presented in the bar graphs. (H) Representative IHC images show reduced IL-21 expression in atherosclerotic lesions of mice following HDCA treatment (magnification, 5 × ; scale bar, 50 μm). (I) Plasma cholesterol levels were measured by enzymatic colorimetry assay in FXR-sufficient controls and FXR KO mice ± HDCA. (J) Plasma triglyceride levels in the Vector and FXR KO groups were quantified at 0, 6, 12, and 18 h by a colorimetric assay. (K) Hemodynamic profiling assessed heart rate, systolic blood pressure (SBP), and diastolic blood pressure (DBP) for each group. (L – M) Ratios of monounsaturated to saturated fatty acids (MUFA/SFA) and polyunsaturated to saturated fatty acids (PUFA/SFA) in serum cholesterol esters (CE) and triglycerides (TG) were quantified by lipidomics in FXR-sufficient control and FXR KO mice ± HDCA. Data are presented as the mean ± SD (n = 3-5 biological replicates). ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: Proteins were detected using the following antibodies: anti-CPT1a antibody (ab234111, abcam), anti-beta actin antibody (ab8226, abcam), anti-PERK antibody (ab229912, abcam), anti-ERK1+ERK2 antibody (ab184699, abcam), anti-S6K1 antibody (ab14708, abcam), anti-S6K1 (phospho T229) antibody (ab5231, abcam), Rac1/2/3 antibody (G-2) (sc-514583, Santa Cruz), anti-Calpain 1 antibody (ab108400, abcam), anti-MMP2 antibody (ab92536, abcam), anti-IL-10 antibody (ab310329, abcam), anti-ZNF671 antibody (HPA046099, Sigma-Aldrich), anti-MAPK6/ERK3 antibody (ab53277, abcam), SIAH1 recombinant rabbit monoclonal antibody (PSH01-80) (MA5-51926, Thermo Fisher), p-Stat1 antibody (pY701.4A) (sc-136229, Santa Cruz), Stat1 antibody (C-136) (sc-464, Santa Cruz), anti-FXR1 antibody (ab155124, abcam), phospho-Raptor (Ser792) polyclonal antibody (PA5-118730, Thermo Fisher), anti-PD1 antibody (ab214421, abcam), SHP-2 antibody (3752S, Cell Signaling Technology), IL-10R antibody (3F9) (sc-53654, Santa Cruz), GAPDH antibody (6C5) (sc-32233, Santa Cruz), rabbit anti-mouse IgG H&L (HRP) (ab6728, abcam), goat anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody, Alexa FluorTM Plus 488 (A32731, Thermo Fisher).

Techniques: In Vivo, Real-time Polymerase Chain Reaction, Expressing, Functional Assay, Flux Assay, Labeling, Western Blot, Clinical Proteomics, Colorimetric Assay, Plasmid Preparation, Control

Journal: The Journal of Experimental Medicine

Article Title: IFNγ-induced memory in human macrophages is sustained by the durability of cytokine signaling itself

doi: 10.1084/jem.20250976

Figure Lengend Snippet: Ruxolitinib blocks LPS and IFNγ-induced Janus kinase signaling. (A) Human macrophages were pre-treated with increasing concentrations of ruxolitinib for 15 min and subsequently stimulated with IFNγ (100 ng/ml) or LPS (100 ng/ml) for 3 h. Whole-cell lysate western blots showing effect of ruxolitinib on STAT1 and STAT2 phosphorylation by each stimulus. Blot is representative of two replicates from separate human donors. (B and C) Quantification of pSTAT2 and (C) pSTAT1 band intensities in A. Source data are available for this figure: .

Article Snippet: The following primary antibodies were used: pSTAT1 pY701.4A (#136229; Santa Cruz Biotechnology, RRID:AB_2019074) diluted at 1:10,000, IRF1 D5E4 (#8478; Cell Signaling Technologies, RRID:AB_10949108) diluted at 1:1,000, β-tubulin TUB2.1 (T5201; Sigma-Aldrich, RRID:AB_609915) diluted at 1:10,000, and GAPDH H-12 (#166574; Santa Cruz Biotechnology, RRID:AB_2107296) diluted at 1:10,000.

Techniques: Western Blot, Phospho-proteomics

Journal: The Journal of Experimental Medicine

Article Title: IFNγ-induced memory in human macrophages is sustained by the durability of cytokine signaling itself

doi: 10.1084/jem.20250976

Figure Lengend Snippet: IFNγ induces long-lasting transcription factor activity and chromatin accessibility after washout. Macrophages were treated with LPS, IFNγ, and LPS in the presence of ruxolitinib for 8 h, as in . Cells were washed and cultured for an additional 88 h. ATACseq was performed after 8 h of stimulation and 4 days after washout. (A) Heatmap of Z-scored reads within ATAC peaks induced by either LPS or IFNγ (L2FC > 2, FDR < 0.01). Clusters were generated by unsupervised k-means clustering. Each column represents a biological replicate from the same human donor. (B) Top enriched motifs in clusters from A. (C) Boxplot quantifying log2 cpm of reads within IFNγ-induced ATAC peaks before and after cytokine washout. (D) Boxplot quantifying log2 cpm of reads within LPS-induced ATAC peaks before and after cytokine washout. (E) Boxplot quantifying log2 cpm of reads within ATAC peaks induced by both IFNγ and LPS (L2FC > 2, FDR < 0.01 for each) peaks before and after cytokine washout. (F) Barplot quantifying percent of transcription factor-bound motifs within STAT1 and IRF1 (IFNγ) and IRF1 and NF-κB (LPS) within induced ATAC peaks in C and D for unstimulated, IFNγ/LPS-stimulated macrophages, and stimulated macrophages 4 days after washout. Motif binding predicted using TOBIAS ATACseq footprinting analysis. Results are average of two technical replicates from a single subject; error bars display standard deviation. (G) Human macrophages were stimulated with IFNγ (100 ng/ml), LPS (100 ng/ml), or IFNβ (10 ng/ml) for 8 h, washed, and then cultured for an additional 66 h. Cells were collected, and whole cell western blotting for phosphorylated STAT1 was performed at the indicated time points. Blot is representative of three replicates from two separate human donors. All box/whisker plots indicate interquartile range and 1.5× interquartile range. Statistical tests were determined by paired Wilcoxon test. ****P < 0.0001. Source data are available for this figure: .

Article Snippet: The following primary antibodies were used: pSTAT1 pY701.4A (#136229; Santa Cruz Biotechnology, RRID:AB_2019074) diluted at 1:10,000, IRF1 D5E4 (#8478; Cell Signaling Technologies, RRID:AB_10949108) diluted at 1:1,000, β-tubulin TUB2.1 (T5201; Sigma-Aldrich, RRID:AB_609915) diluted at 1:10,000, and GAPDH H-12 (#166574; Santa Cruz Biotechnology, RRID:AB_2107296) diluted at 1:10,000.

Techniques: Activity Assay, Cell Culture, Generated, Binding Assay, Footprinting, Standard Deviation, Western Blot, Whisker Assay

Journal: The Journal of Experimental Medicine

Article Title: IFNγ-induced memory in human macrophages is sustained by the durability of cytokine signaling itself

doi: 10.1084/jem.20250976

Figure Lengend Snippet: Cell surface–bound IFNγ mediates persistent signaling regardless of cytokine manufacturer and can be degraded by trypsinization. (A) Quantification of pSTAT1 intensity normalized to 3H IFNγ in . (B) Human macrophages were treated with Escherichia coli sourced IFNγ purchased from PeproTech and mammalian-sourced IFNγ purchased from Sigma-Aldrich and ACRO. Cells were treated at 100 ng/ml for 8 h, washed, and cultured for an additional 48 h in regular media prior to collection. Cells were collected for immunoblot at the indicated times. Representative blot of duplicates from one human subject. (C) Quantification of pSTAT1 band intensity normalized to tubulin at each time point in B. (D) Human A549 airway epithelial cells were stimulated with 100 ng/ml IFNγ, washed, and cultured for an additional 72 h. Cells were collected for immunoblot at the indicated timepoints. Representative blot of two replicates. (E) Quantification of pSTAT1 band intensity normalized to tubulin at each time point in D. (F) Human macrophages were treated with 100 ng/ml IFNγ for 8 h, washed, and lifted by scraping after incubation with either PBS, 0.5 mM EDTA in PBS, or trypsin. Cells were replated and cultured for an additional 24 h in regular media. Cells were collected for immunoblot at the indicated times. (G) Quantification of pSTAT1 band intensity normalized to GAPDH for each condition in F. Statistical tests were determined by single-tailed t test. *P < 0.05; **P < 0.01. Source data are available for this figure: .

Article Snippet: The following primary antibodies were used: pSTAT1 pY701.4A (#136229; Santa Cruz Biotechnology, RRID:AB_2019074) diluted at 1:10,000, IRF1 D5E4 (#8478; Cell Signaling Technologies, RRID:AB_10949108) diluted at 1:1,000, β-tubulin TUB2.1 (T5201; Sigma-Aldrich, RRID:AB_609915) diluted at 1:10,000, and GAPDH H-12 (#166574; Santa Cruz Biotechnology, RRID:AB_2107296) diluted at 1:10,000.

Techniques: Cell Culture, Western Blot, Incubation

Journal: The Journal of Experimental Medicine

Article Title: IFNγ-induced memory in human macrophages is sustained by the durability of cytokine signaling itself

doi: 10.1084/jem.20250976

Figure Lengend Snippet: Cell surface–bound IFNγ mediates persistent JAK/STAT signaling even after cytokine washout. (A) Human macrophages were stimulated with IFNγ (100 ng/ml) for 8 h, washed, and then cultured in regular media or media containing ruxolitinib (1 µM) or increasing concentrations of anti-IFNγ neutralizing antibody for an additional 28 h. Cells were collected, and whole cell western blotting for phosphorylated STAT1 and IRF1 was performed at indicated time points. Representative blot of duplicates from two separate subjects. (B) Human macrophages were stimulated with 100 ng/ml IFNγ for 3 h, washed, and then cultured in either ruxolitinib (1 µM), anti-IFNγ neutralizing antibody (10 µg/ml), or isotype control antibody (10 µg/ml) for 2 h. Samples were collected at the indicated times for immunoblot. Representative blot of duplicates from two separate subjects. (C) Quantification of pSTAT1 band intensities from B. (D) Human macrophages were stimulated with 100 ng/ml IFNγ for 8 h, washed, and cultured for an additional 88 h in regular media. Supernatants from stimulated macrophages were collected after the 8-h stimulation and 88 h after washout. This supernatant was used to stimulate fresh macrophages for 1 h in the presence/absence of ruxolitinib (1 µM) or anti-IFNγ neutralizing antibody (10 µg/ml). As a control, fresh macrophages were stimulated with media supplemented with 1 ng/ml IFNγ for 1 h. Representative blot of duplicates from two separate subjects. (E) Macrophages were left in regular media or pre-treated with 10 µg/ml CHX for 15 min and stimulated with 100 ng/ml IFNγ for 3 h. Treated macrophages were washed and subsequently cultured for 2 h in regular media, media supplemented with 10 µg/ml CHX, or anti-IFNγ neutralizing antibody (10 µg/ml) and collected for immunoblot. Duplicates from one subject are shown. (F) Quantification of pSTAT1 band intensities in E normalized to band intensity of macrophages treated with IFNγ for 3 h. (G) Human macrophages were stimulated with 100 ng/ml IFNγ for 8 h, washed, and cultured in regular media or media supplemented with 1 µM ruxolitinib for 16 h. After 16 h, cells were washed again and cultured in regular media for an additional 24 h. Cells were collected for immunoblot at indicated times. Representative blot of four replicates from two subjects. (H) Quantification of pSTAT1 band intensities in G normalized to band intensity of macrophages treated with IFNγ for 3 h. Statistical tests were determined by a single-tailed t test. *P < 0.05, **P < 0.01, and ***P < 0.001. Source data are available for this figure: .

Article Snippet: The following primary antibodies were used: pSTAT1 pY701.4A (#136229; Santa Cruz Biotechnology, RRID:AB_2019074) diluted at 1:10,000, IRF1 D5E4 (#8478; Cell Signaling Technologies, RRID:AB_10949108) diluted at 1:1,000, β-tubulin TUB2.1 (T5201; Sigma-Aldrich, RRID:AB_609915) diluted at 1:10,000, and GAPDH H-12 (#166574; Santa Cruz Biotechnology, RRID:AB_2107296) diluted at 1:10,000.

Techniques: Cell Culture, Western Blot, Control

Journal: Molecular Medicine Reports

Article Title: STAT1 accelerates cutaneous melanoma progression through TUBB4A expression regulation

doi: 10.3892/mmr.2026.13828

Figure Lengend Snippet: STAT1 and TUBB4A expression levels are significantly correlated and upregulated in melanoma. (A) Analysis of data in the Gene Expression Profiling Interactive Analysis 2 database revealed a significant elevation of STAT1 expression in melanoma tissues. Quantification of (B) STAT1 and (C) TUBB4A mRNA levels in 31 paired melanoma and normal tissue samples from patients with SKCM demonstrated a significant upregulation of both STAT1 and TUBB4A expression in melanoma tissues. (D) Correlation analysis of STAT1 and TUBB4A mRNA levels in melanoma samples showed a significant positive correlation. Comparative analysis of (E) STAT1 and (F) TUBB4A mRNA expression in normal HEM and melanoma cell lines revealed significantly higher expression in the melanoma cell lines. *P<0.05 between groups; **P<0.01 compared with HEM cells. HEM, human epidermal melanocytes; STAT1, signal transducer and activator of transcription 1; TUBB4A, tubulin β4A; TPM, transcripts per million; SKCM, skin cutaneous melanoma.

Article Snippet: For each reaction, 100 μg chromatin lysate was diluted to a final volume of 500 μl with ChIP dilution buffer and incubated with 5 μg of anti-STAT1 antibodies (1:100; cat. no. 14994; Cell Signaling Technology, Inc.) or control rabbit IgG (1:100; cat. no. 2729; Cell Signaling Technology, Inc.) overnight at 4°C.

Techniques: Expressing, Gene Expression

Journal: Molecular Medicine Reports

Article Title: STAT1 accelerates cutaneous melanoma progression through TUBB4A expression regulation

doi: 10.3892/mmr.2026.13828

Figure Lengend Snippet: STAT1 knockdown significantly reduces melanoma cell viability and proliferation. STAT1 mRNA expression was analyzed in (A) A375 and (B) RPMI-7951 melanoma cells post-STAT1 knockdown using siRNA. (C) Western blotting was used to assess STAT1 protein levels in melanoma cell lines following siRNA-mediated knockdown. Cell viability was evaluated after STAT1 knockdown in (D) A375 and (E) RPMI-7951 cells using Cell Counting Kit-8 assays. (F) Colony formation assays were performed to assess the proliferative capacity of cells after STAT1 knockdown. (G) Quantification of colony formation assay results. **P<0.01 vs. si-NC group. siRNA, small interfering RNA; STAT1, signal transducer and activator of transcription 1; si-STAT1, siRNA targeting STAT1; si-NC, negative control siRNA.

Article Snippet: For each reaction, 100 μg chromatin lysate was diluted to a final volume of 500 μl with ChIP dilution buffer and incubated with 5 μg of anti-STAT1 antibodies (1:100; cat. no. 14994; Cell Signaling Technology, Inc.) or control rabbit IgG (1:100; cat. no. 2729; Cell Signaling Technology, Inc.) overnight at 4°C.

Techniques: Knockdown, Expressing, Western Blot, Cell Counting, Colony Assay, Small Interfering RNA, Negative Control

Journal: Molecular Medicine Reports

Article Title: STAT1 accelerates cutaneous melanoma progression through TUBB4A expression regulation

doi: 10.3892/mmr.2026.13828

Figure Lengend Snippet: STAT1 knockdown significantly promotes melanoma cell apoptosis and inhibits migration. (A and B) Apoptosis was assessed and quantified in A375 and RPMI-7951 cells following STAT1 knockdown using flow cytometry. (C) Transwell migration assays were conducted to evaluate cell migration following STAT1 knockdown. Magnification, ×200. (D) Quantification of migration capacity in A375 and RPMI-7951 cells following STAT1 knockdown. *P<0.05 vs. si-NC. STAT1, signal transducer and activator of transcription 1; si-STAT1, small interfering RNA targeting STAT1; si-NC, negative control small interfering RNA.

Article Snippet: For each reaction, 100 μg chromatin lysate was diluted to a final volume of 500 μl with ChIP dilution buffer and incubated with 5 μg of anti-STAT1 antibodies (1:100; cat. no. 14994; Cell Signaling Technology, Inc.) or control rabbit IgG (1:100; cat. no. 2729; Cell Signaling Technology, Inc.) overnight at 4°C.

Techniques: Knockdown, Migration, Flow Cytometry, Small Interfering RNA, Negative Control

Journal: Molecular Medicine Reports

Article Title: STAT1 accelerates cutaneous melanoma progression through TUBB4A expression regulation

doi: 10.3892/mmr.2026.13828

Figure Lengend Snippet: STAT1 regulates TUBB4A expression at the transcription level. (A) STAT1 mRNA levels were measured in A375 and RPMI-7951 cells after STAT1 knockdown via transfection with different siRNA sequences. (B) TUBB4A mRNA levels were measured in A375 and RPMI-7951 cells after STAT1 knockdown via transfection with different siRNA sequences. (C) Specific fragments of the TUBB4A promoter region were cloned into the luciferase reporter plasmids upstream of the firefly luciferase gene. (D) Transcriptional activity of various TUBB4A promoter fragments was analyzed by luciferase reporter assay in 293T cells, with the −1,783 and −1,771 fragments exhibiting the highest activity. (E) STAT1 siRNA-mediated knockdown significantly reduced STAT1 mRNA levels in A375 cells. (F) STAT1 knockdown significantly reduced the luciferase activity of the −1,783 fragment of the TUBB4A promoter, but not the −1,771 fragment. (G) Chromatin immunoprecipitation assays were performed in A375 and RPMI-7951 cells targeting the −1,783 binding site in the TUBB4A promoter region. Quantitative PCR provided evidence of STAT1 binding to this region. Genomic DNA input was set to 100%. **P<0.01 vs. si-NC; ## P<0.01 vs. PGL3; && P<0.01 vs. IgG. STAT1, signal transducer and activator of transcription 1; siRNA, small interfering RNA; si-NC, negative control siRNA; si-STAT1, siRNA targeting STAT1; si-STAT1-1, siRNA targeting STAT1 sequence 1; si-STAT1-2, siRNA targeting STAT1 sequence 2; TUBB4A, tubulin β4A; PGL3, promoter-gluc luciferase 3; LUC, firefly luciferase gene.

Article Snippet: For each reaction, 100 μg chromatin lysate was diluted to a final volume of 500 μl with ChIP dilution buffer and incubated with 5 μg of anti-STAT1 antibodies (1:100; cat. no. 14994; Cell Signaling Technology, Inc.) or control rabbit IgG (1:100; cat. no. 2729; Cell Signaling Technology, Inc.) overnight at 4°C.

Techniques: Expressing, Knockdown, Transfection, Clone Assay, Luciferase, Activity Assay, Reporter Assay, Chromatin Immunoprecipitation, Binding Assay, Real-time Polymerase Chain Reaction, Small Interfering RNA, Negative Control, Sequencing

Journal: Molecular Medicine Reports

Article Title: STAT1 accelerates cutaneous melanoma progression through TUBB4A expression regulation

doi: 10.3892/mmr.2026.13828

Figure Lengend Snippet: TUBB4A overexpression mitigates the effects of STAT1 knockdown on cell viability and proliferation. TUBB4A mRNA levels were measured in (A) A375 and (B) RPMI-7951 cells following TUBB4A overexpression mediated by a lentiviral vector. (C) TUBB4A protein expression was analyzed after its overexpression. Combined STAT1 knockdown and TUBB4A overexpression transfections were performed, followed by a western blot analysis of TUBB4A protein levels in (D) A375 and (E) RPMI-7951 cells. Cell viability was assessed via Cell Counting Kit-8 assays following combined STAT1 knockdown and TUBB4A overexpression in (F) A375 and (G) RPMI-7951 cells. (H) Colony formation assays were performed to assess the proliferative capacity of cells after STAT1 knockdown and TUBB4A overexpression. (I) Quantification of colony formation assay results. **P<0.01 vs. Ov-NC; ## P<0.01 vs. si-STAT1. TUBB4A, tubulin β4A; STAT1, signal transducer and activator of transcription 1; si-NC, negative control small interfering RNA; si-STAT1, small interfering RNA targeting STAT1; Ov-NC, negative control lentiviral overexpression vector; Ov-TUBB4A, lentiviral vector for TUBB4A overexpression.

Article Snippet: For each reaction, 100 μg chromatin lysate was diluted to a final volume of 500 μl with ChIP dilution buffer and incubated with 5 μg of anti-STAT1 antibodies (1:100; cat. no. 14994; Cell Signaling Technology, Inc.) or control rabbit IgG (1:100; cat. no. 2729; Cell Signaling Technology, Inc.) overnight at 4°C.

Techniques: Over Expression, Knockdown, Plasmid Preparation, Expressing, Transfection, Western Blot, Cell Counting, Colony Assay, Negative Control, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: STAT1 accelerates cutaneous melanoma progression through TUBB4A expression regulation

doi: 10.3892/mmr.2026.13828

Figure Lengend Snippet: TUBB4A overexpression reverses the effects of STAT1 knockdown on apoptosis, migration and tumor growth. (A-D) Apoptosis and migration were evaluated in A375 and RPMI-7951 cells following STAT1 knockdown and TUBB4A overexpression. Magnification, ×200. (E) Representative images of isolated xenograft tumors in mice. Tumor volumes were measured in nude mice injected subcutaneously with 2×10 6 A375 cells that had been subject to STAT1 knockdown and TUBB4A overexpression. (F) Quantification of mouse tumor volumes showed that STAT1 knockdown significantly suppressed tumor growth, whereas TUBB4A overexpression reversed this inhibitory effect. ## P<0.01 vs. si-STAT1. TUBB4A, tubulin β4A; si-NC, negative control small interfering RNA; Ov-TUBB4A, lentiviral vector for TUBB4A overexpression.

Article Snippet: For each reaction, 100 μg chromatin lysate was diluted to a final volume of 500 μl with ChIP dilution buffer and incubated with 5 μg of anti-STAT1 antibodies (1:100; cat. no. 14994; Cell Signaling Technology, Inc.) or control rabbit IgG (1:100; cat. no. 2729; Cell Signaling Technology, Inc.) overnight at 4°C.

Techniques: Over Expression, Knockdown, Migration, Isolation, Injection, Negative Control, Small Interfering RNA, Plasmid Preparation

Journal: Journal of Advanced Research

Article Title: Transglutaminase 2 modulates inflammatory angiogenesis via vascular endothelial growth factor receptor 2 pathway in inflammatory bowel disease

doi: 10.1016/j.jare.2025.07.002

Figure Lengend Snippet: STAT1-TGM2-VEGFR2 axis drives inflammatory angiogenesis in IBD. (A) The mRNA transcription of TGM2 was significantly upregulated upon stimulation with inflammatory cytokines IL-9, IL-23, and IFN-γ in HIMECs (n = 3 in each group). (B, C) The results of WB demonstrated that stimulation with IL-9, IL-23, and IFN-γ upregulated the protein expression of TGM2 in HIMEC. (D) Stimulation with inflammatory cytokines IL-9, IL-23, and IFN-γ significantly augmented the in vitro angiogenic capacity of HIMECs, while knockdown of TGM2 effectively reversed this effect. (E, F) IL-9, IL-23 and IFN-γ stimulation significantly enhanced the phosphorylation of VEGFR2 at Tyr1059, Tyr1214 and activated the downstream pathways such as FAK, PLC-γ and MAPK pathways in HIMEC, which were reversed by TGM2 knockdown. (G) IF results showed VEGFR2 co-localizes with TGM2 in HIMEC, mainly on the cell membrane. (H) Co-IP results substantiated the interaction between TGM2 and VEGFR2. (I) The CHIP assay confirmed the binding of STAT1 to the upstream promoter region of TGM2, thereby facilitating transcription following IFN-γ stimulation. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: The antibody-protein complexes were immunoprecipitated using an anti-p-STAT1 antibody (9107, CST, USA) and control IgG.

Techniques: Expressing, In Vitro, Knockdown, Phospho-proteomics, Membrane, Co-Immunoprecipitation Assay, Binding Assay

Journal: Molecular Medicine Reports

Article Title: STAT1 accelerates cutaneous melanoma progression through TUBB4A expression regulation

doi: 10.3892/mmr.2026.13828

Figure Lengend Snippet: STAT1 and TUBB4A expression levels are significantly correlated and upregulated in melanoma. (A) Analysis of data in the Gene Expression Profiling Interactive Analysis 2 database revealed a significant elevation of STAT1 expression in melanoma tissues. Quantification of (B) STAT1 and (C) TUBB4A mRNA levels in 31 paired melanoma and normal tissue samples from patients with SKCM demonstrated a significant upregulation of both STAT1 and TUBB4A expression in melanoma tissues. (D) Correlation analysis of STAT1 and TUBB4A mRNA levels in melanoma samples showed a significant positive correlation. Comparative analysis of (E) STAT1 and (F) TUBB4A mRNA expression in normal HEM and melanoma cell lines revealed significantly higher expression in the melanoma cell lines. *P<0.05 between groups; **P<0.01 compared with HEM cells. HEM, human epidermal melanocytes; STAT1, signal transducer and activator of transcription 1; TUBB4A, tubulin β4A; TPM, transcripts per million; SKCM, skin cutaneous melanoma.

Article Snippet: Membranes were blocked with 5% non-fat milk (cat. no. 1706404; Bio-Rad Laboratories, Inc.) in TBST with 0.1% Tween 20 for 1 h at 25°C and incubated overnight at 4°C with primary antibodies against STAT1 (1:1,000; cat. no. 14994; Cell Signaling Technology, Inc.) and TUBB4A (1:1,000; cat. no. ab11315; Abcam), as well as GAPDH (1:5,000; cat. no. 2118; Cell Signaling Technology, Inc.) serving as a loading control.

Techniques: Expressing, Gene Expression

Journal: Molecular Medicine Reports

Article Title: STAT1 accelerates cutaneous melanoma progression through TUBB4A expression regulation

doi: 10.3892/mmr.2026.13828

Figure Lengend Snippet: STAT1 knockdown significantly reduces melanoma cell viability and proliferation. STAT1 mRNA expression was analyzed in (A) A375 and (B) RPMI-7951 melanoma cells post-STAT1 knockdown using siRNA. (C) Western blotting was used to assess STAT1 protein levels in melanoma cell lines following siRNA-mediated knockdown. Cell viability was evaluated after STAT1 knockdown in (D) A375 and (E) RPMI-7951 cells using Cell Counting Kit-8 assays. (F) Colony formation assays were performed to assess the proliferative capacity of cells after STAT1 knockdown. (G) Quantification of colony formation assay results. **P<0.01 vs. si-NC group. siRNA, small interfering RNA; STAT1, signal transducer and activator of transcription 1; si-STAT1, siRNA targeting STAT1; si-NC, negative control siRNA.

Article Snippet: Membranes were blocked with 5% non-fat milk (cat. no. 1706404; Bio-Rad Laboratories, Inc.) in TBST with 0.1% Tween 20 for 1 h at 25°C and incubated overnight at 4°C with primary antibodies against STAT1 (1:1,000; cat. no. 14994; Cell Signaling Technology, Inc.) and TUBB4A (1:1,000; cat. no. ab11315; Abcam), as well as GAPDH (1:5,000; cat. no. 2118; Cell Signaling Technology, Inc.) serving as a loading control.

Techniques: Knockdown, Expressing, Western Blot, Cell Counting, Colony Assay, Small Interfering RNA, Negative Control

Journal: Molecular Medicine Reports

Article Title: STAT1 accelerates cutaneous melanoma progression through TUBB4A expression regulation

doi: 10.3892/mmr.2026.13828

Figure Lengend Snippet: STAT1 knockdown significantly promotes melanoma cell apoptosis and inhibits migration. (A and B) Apoptosis was assessed and quantified in A375 and RPMI-7951 cells following STAT1 knockdown using flow cytometry. (C) Transwell migration assays were conducted to evaluate cell migration following STAT1 knockdown. Magnification, ×200. (D) Quantification of migration capacity in A375 and RPMI-7951 cells following STAT1 knockdown. *P<0.05 vs. si-NC. STAT1, signal transducer and activator of transcription 1; si-STAT1, small interfering RNA targeting STAT1; si-NC, negative control small interfering RNA.

Article Snippet: Membranes were blocked with 5% non-fat milk (cat. no. 1706404; Bio-Rad Laboratories, Inc.) in TBST with 0.1% Tween 20 for 1 h at 25°C and incubated overnight at 4°C with primary antibodies against STAT1 (1:1,000; cat. no. 14994; Cell Signaling Technology, Inc.) and TUBB4A (1:1,000; cat. no. ab11315; Abcam), as well as GAPDH (1:5,000; cat. no. 2118; Cell Signaling Technology, Inc.) serving as a loading control.

Techniques: Knockdown, Migration, Flow Cytometry, Small Interfering RNA, Negative Control

Journal: Molecular Medicine Reports

Article Title: STAT1 accelerates cutaneous melanoma progression through TUBB4A expression regulation

doi: 10.3892/mmr.2026.13828

Figure Lengend Snippet: STAT1 regulates TUBB4A expression at the transcription level. (A) STAT1 mRNA levels were measured in A375 and RPMI-7951 cells after STAT1 knockdown via transfection with different siRNA sequences. (B) TUBB4A mRNA levels were measured in A375 and RPMI-7951 cells after STAT1 knockdown via transfection with different siRNA sequences. (C) Specific fragments of the TUBB4A promoter region were cloned into the luciferase reporter plasmids upstream of the firefly luciferase gene. (D) Transcriptional activity of various TUBB4A promoter fragments was analyzed by luciferase reporter assay in 293T cells, with the −1,783 and −1,771 fragments exhibiting the highest activity. (E) STAT1 siRNA-mediated knockdown significantly reduced STAT1 mRNA levels in A375 cells. (F) STAT1 knockdown significantly reduced the luciferase activity of the −1,783 fragment of the TUBB4A promoter, but not the −1,771 fragment. (G) Chromatin immunoprecipitation assays were performed in A375 and RPMI-7951 cells targeting the −1,783 binding site in the TUBB4A promoter region. Quantitative PCR provided evidence of STAT1 binding to this region. Genomic DNA input was set to 100%. **P<0.01 vs. si-NC; ## P<0.01 vs. PGL3; && P<0.01 vs. IgG. STAT1, signal transducer and activator of transcription 1; siRNA, small interfering RNA; si-NC, negative control siRNA; si-STAT1, siRNA targeting STAT1; si-STAT1-1, siRNA targeting STAT1 sequence 1; si-STAT1-2, siRNA targeting STAT1 sequence 2; TUBB4A, tubulin β4A; PGL3, promoter-gluc luciferase 3; LUC, firefly luciferase gene.

Article Snippet: Membranes were blocked with 5% non-fat milk (cat. no. 1706404; Bio-Rad Laboratories, Inc.) in TBST with 0.1% Tween 20 for 1 h at 25°C and incubated overnight at 4°C with primary antibodies against STAT1 (1:1,000; cat. no. 14994; Cell Signaling Technology, Inc.) and TUBB4A (1:1,000; cat. no. ab11315; Abcam), as well as GAPDH (1:5,000; cat. no. 2118; Cell Signaling Technology, Inc.) serving as a loading control.

Techniques: Expressing, Knockdown, Transfection, Clone Assay, Luciferase, Activity Assay, Reporter Assay, Chromatin Immunoprecipitation, Binding Assay, Real-time Polymerase Chain Reaction, Small Interfering RNA, Negative Control, Sequencing

Journal: Molecular Medicine Reports

Article Title: STAT1 accelerates cutaneous melanoma progression through TUBB4A expression regulation

doi: 10.3892/mmr.2026.13828

Figure Lengend Snippet: TUBB4A overexpression mitigates the effects of STAT1 knockdown on cell viability and proliferation. TUBB4A mRNA levels were measured in (A) A375 and (B) RPMI-7951 cells following TUBB4A overexpression mediated by a lentiviral vector. (C) TUBB4A protein expression was analyzed after its overexpression. Combined STAT1 knockdown and TUBB4A overexpression transfections were performed, followed by a western blot analysis of TUBB4A protein levels in (D) A375 and (E) RPMI-7951 cells. Cell viability was assessed via Cell Counting Kit-8 assays following combined STAT1 knockdown and TUBB4A overexpression in (F) A375 and (G) RPMI-7951 cells. (H) Colony formation assays were performed to assess the proliferative capacity of cells after STAT1 knockdown and TUBB4A overexpression. (I) Quantification of colony formation assay results. **P<0.01 vs. Ov-NC; ## P<0.01 vs. si-STAT1. TUBB4A, tubulin β4A; STAT1, signal transducer and activator of transcription 1; si-NC, negative control small interfering RNA; si-STAT1, small interfering RNA targeting STAT1; Ov-NC, negative control lentiviral overexpression vector; Ov-TUBB4A, lentiviral vector for TUBB4A overexpression.

Article Snippet: Membranes were blocked with 5% non-fat milk (cat. no. 1706404; Bio-Rad Laboratories, Inc.) in TBST with 0.1% Tween 20 for 1 h at 25°C and incubated overnight at 4°C with primary antibodies against STAT1 (1:1,000; cat. no. 14994; Cell Signaling Technology, Inc.) and TUBB4A (1:1,000; cat. no. ab11315; Abcam), as well as GAPDH (1:5,000; cat. no. 2118; Cell Signaling Technology, Inc.) serving as a loading control.

Techniques: Over Expression, Knockdown, Plasmid Preparation, Expressing, Transfection, Western Blot, Cell Counting, Colony Assay, Negative Control, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: STAT1 accelerates cutaneous melanoma progression through TUBB4A expression regulation

doi: 10.3892/mmr.2026.13828

Figure Lengend Snippet: TUBB4A overexpression reverses the effects of STAT1 knockdown on apoptosis, migration and tumor growth. (A-D) Apoptosis and migration were evaluated in A375 and RPMI-7951 cells following STAT1 knockdown and TUBB4A overexpression. Magnification, ×200. (E) Representative images of isolated xenograft tumors in mice. Tumor volumes were measured in nude mice injected subcutaneously with 2×10 6 A375 cells that had been subject to STAT1 knockdown and TUBB4A overexpression. (F) Quantification of mouse tumor volumes showed that STAT1 knockdown significantly suppressed tumor growth, whereas TUBB4A overexpression reversed this inhibitory effect. ## P<0.01 vs. si-STAT1. TUBB4A, tubulin β4A; si-NC, negative control small interfering RNA; Ov-TUBB4A, lentiviral vector for TUBB4A overexpression.

Article Snippet: Membranes were blocked with 5% non-fat milk (cat. no. 1706404; Bio-Rad Laboratories, Inc.) in TBST with 0.1% Tween 20 for 1 h at 25°C and incubated overnight at 4°C with primary antibodies against STAT1 (1:1,000; cat. no. 14994; Cell Signaling Technology, Inc.) and TUBB4A (1:1,000; cat. no. ab11315; Abcam), as well as GAPDH (1:5,000; cat. no. 2118; Cell Signaling Technology, Inc.) serving as a loading control.

Techniques: Over Expression, Knockdown, Migration, Isolation, Injection, Negative Control, Small Interfering RNA, Plasmid Preparation

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Therapeutic effect of eravacycline against carbapenem-resistant hypervirulent Klebsiella pneumoniae in mouse models

doi: 10.1128/aac.01237-25

Figure Lengend Snippet: Expression and activation of STAT1 in mouse lung tissues. ( A ) mRNA expression levels of STAT1 in the lung tissues of mice across the uninfected control, the infection control, and the treatment groups. ( B ) Protein expression levels of p-STAT1 in the uninfected control, the infection control, and the treatment groups of mice. Data are shown as mean ± standard error of the mean, n = 3. Hashtags (#) represent the infection control group compared to the uninfected control group, and asterisks (*) represent the infection control group compared to the corresponding treatment group (* P < 0.05, ** P < 0.01, and ### P < 0.001).

Article Snippet: The membrane was blocked with TBST buffer (G2150, Servicebio Technology Co., Ltd. Wuhan, China) containing 5% bovine serum albumin and then incubated with primary antibody STAT1 (1:1,000, #9172T, Cell Signaling Technology, MA, USA), p-STAT1 (1:1,000, #7649T, Cell Signaling Technology, MA, USA), and β-actin (1:10,000, AC004, Abclonal, Wuhan, China) overnight at 4°C.

Techniques: Expressing, Activation Assay, Control, Infection