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: 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: 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

Journal: Chinese Medicine

Article Title: Matrine improves bile acid metabolism and reduces inflammatory and oxidative stress in colitis via the JAK2 pathway

doi: 10.1186/s13020-026-01387-z

Figure Lengend Snippet: Effects of matrine on intestinal epithelial cell function upon inflammation Mouse intestinal epithelial cell line, MODE-K, was stimulated with LPS with or without matrine treatment (1, 2, 3 mg/ml), and examined for the protein levels of IL-1β, TNF-α, and IL-6 using Immunoblotting ( A ); The levels of MPO, NO, and MDA using commercial kits ( B ); Intracellular ROS levels were detected by flow cytometry ( C ); The protein levels of FXR, MRP3 and MRP4 were examined using Immunoblotting ( D ); The mRNA levels of MRP3, MRP4, OSTα and OSTβ were examined using qRT-PCR ( E ); The protein levels of p-JAK2, JAK2, p-STAT3, STAT3, p-STAT1, and STAT1 using Immunoblotting ( F ). **p < 0.01, vs. PBS group; #p < 0.05, ##p < 0.01 vs. LPS group

Article Snippet: Membrane was blocked within Odyssey blocking buffer (LI-COR Bioscience, Lincoln, USA) for 1 h at room temperature (RT), and then incubated with primary antibodies against p-JAK2 (AP0917, Abclonal, Woburn, USA), JAK2 (AF6022, Affinitiy Bioscience, Changzhou, China), MRP3 (bs-0656R, Bioss, Beijing, China), MRP4 (DF6921, Affinity Bioscience), IL-1β [12242, Cell Signaling Technology (CST), Danvers, USA], TNF-α (11948, CST), IL-6 (CSB-PA06757A0RB, CUSABIO, Wuhan, China), p-STAT3 (AF3293, Affinity Bioscience), STAT3 (10253-2-AP, Proteintech, Wuhan, China), p-STAT1 (28977-1-AP, Protientech), STAT1 (10144-2-AP, Proteintech), FXR (M022312, Abmart, Shanghai, China), and GAPDH (endogenous control, 60004-1-Ig, Proteintech) overnight at 4 °C (dilution 1:1000).

Techniques: Cell Function Assay, Western Blot, Flow Cytometry, Quantitative RT-PCR

Journal: Chinese Medicine

Article Title: Matrine improves bile acid metabolism and reduces inflammatory and oxidative stress in colitis via the JAK2 pathway

doi: 10.1186/s13020-026-01387-z

Figure Lengend Snippet: Dynamic effects of matrine and JAK2 on intestinal epithelial cell function upon inflammation MODE-K cells were stimulated with LPS with or without matrine treatment (2 mg/ml), transfected with JAK2-overexpressing plasmid (JAK2 oe), and examined for the protein levels of IL-1β, TNF-α, and IL-6 using Immunoblotting ( A ); the levels of MPO, NO, and MDA using commercial kits ( B ); Intracellular ROS levels were detected by flow cytometry ( C ); The protein levels of FXR, MRP3 and MRP4 were examined using Immunoblotting ( D ); The mRNA levels of MRP3, MRP4, OSTα and OSTβ were examined using qRT-PCR ( E ); the protein levels of JAK2, p-STAT3, STAT3, p-STAT1, and STAT1 using Immunoblotting ( F ). **p < 0.01, vs. LPS group; #p < 0.05, ## p < 0.01 vs. LPS + matrine + vector group

Article Snippet: Membrane was blocked within Odyssey blocking buffer (LI-COR Bioscience, Lincoln, USA) for 1 h at room temperature (RT), and then incubated with primary antibodies against p-JAK2 (AP0917, Abclonal, Woburn, USA), JAK2 (AF6022, Affinitiy Bioscience, Changzhou, China), MRP3 (bs-0656R, Bioss, Beijing, China), MRP4 (DF6921, Affinity Bioscience), IL-1β [12242, Cell Signaling Technology (CST), Danvers, USA], TNF-α (11948, CST), IL-6 (CSB-PA06757A0RB, CUSABIO, Wuhan, China), p-STAT3 (AF3293, Affinity Bioscience), STAT3 (10253-2-AP, Proteintech, Wuhan, China), p-STAT1 (28977-1-AP, Protientech), STAT1 (10144-2-AP, Proteintech), FXR (M022312, Abmart, Shanghai, China), and GAPDH (endogenous control, 60004-1-Ig, Proteintech) overnight at 4 °C (dilution 1:1000).

Techniques: Cell Function Assay, Transfection, Plasmid Preparation, Western Blot, Flow Cytometry, Quantitative RT-PCR

Journal: Chinese Medicine

Article Title: Matrine improves bile acid metabolism and reduces inflammatory and oxidative stress in colitis via the JAK2 pathway

doi: 10.1186/s13020-026-01387-z

Figure Lengend Snippet: Effects of matrine on intestinal epithelial cell function upon inflammation Mouse intestinal epithelial cell line, MODE-K, was stimulated with LPS with or without matrine treatment (1, 2, 3 mg/ml), and examined for the protein levels of IL-1β, TNF-α, and IL-6 using Immunoblotting ( A ); The levels of MPO, NO, and MDA using commercial kits ( B ); Intracellular ROS levels were detected by flow cytometry ( C ); The protein levels of FXR, MRP3 and MRP4 were examined using Immunoblotting ( D ); The mRNA levels of MRP3, MRP4, OSTα and OSTβ were examined using qRT-PCR ( E ); The protein levels of p-JAK2, JAK2, p-STAT3, STAT3, p-STAT1, and STAT1 using Immunoblotting ( F ). **p < 0.01, vs. PBS group; #p < 0.05, ##p < 0.01 vs. LPS group

Article Snippet: Membrane was blocked within Odyssey blocking buffer (LI-COR Bioscience, Lincoln, USA) for 1 h at room temperature (RT), and then incubated with primary antibodies against p-JAK2 (AP0917, Abclonal, Woburn, USA), JAK2 (AF6022, Affinitiy Bioscience, Changzhou, China), MRP3 (bs-0656R, Bioss, Beijing, China), MRP4 (DF6921, Affinity Bioscience), IL-1β [12242, Cell Signaling Technology (CST), Danvers, USA], TNF-α (11948, CST), IL-6 (CSB-PA06757A0RB, CUSABIO, Wuhan, China), p-STAT3 (AF3293, Affinity Bioscience), STAT3 (10253-2-AP, Proteintech, Wuhan, China), p-STAT1 (28977-1-AP, Protientech), STAT1 (10144-2-AP, Proteintech), FXR (M022312, Abmart, Shanghai, China), and GAPDH (endogenous control, 60004-1-Ig, Proteintech) overnight at 4 °C (dilution 1:1000).

Techniques: Cell Function Assay, Western Blot, Flow Cytometry, Quantitative RT-PCR

Journal: Chinese Medicine

Article Title: Matrine improves bile acid metabolism and reduces inflammatory and oxidative stress in colitis via the JAK2 pathway

doi: 10.1186/s13020-026-01387-z

Figure Lengend Snippet: Dynamic effects of matrine and JAK2 on intestinal epithelial cell function upon inflammation MODE-K cells were stimulated with LPS with or without matrine treatment (2 mg/ml), transfected with JAK2-overexpressing plasmid (JAK2 oe), and examined for the protein levels of IL-1β, TNF-α, and IL-6 using Immunoblotting ( A ); the levels of MPO, NO, and MDA using commercial kits ( B ); Intracellular ROS levels were detected by flow cytometry ( C ); The protein levels of FXR, MRP3 and MRP4 were examined using Immunoblotting ( D ); The mRNA levels of MRP3, MRP4, OSTα and OSTβ were examined using qRT-PCR ( E ); the protein levels of JAK2, p-STAT3, STAT3, p-STAT1, and STAT1 using Immunoblotting ( F ). **p < 0.01, vs. LPS group; #p < 0.05, ## p < 0.01 vs. LPS + matrine + vector group

Article Snippet: Membrane was blocked within Odyssey blocking buffer (LI-COR Bioscience, Lincoln, USA) for 1 h at room temperature (RT), and then incubated with primary antibodies against p-JAK2 (AP0917, Abclonal, Woburn, USA), JAK2 (AF6022, Affinitiy Bioscience, Changzhou, China), MRP3 (bs-0656R, Bioss, Beijing, China), MRP4 (DF6921, Affinity Bioscience), IL-1β [12242, Cell Signaling Technology (CST), Danvers, USA], TNF-α (11948, CST), IL-6 (CSB-PA06757A0RB, CUSABIO, Wuhan, China), p-STAT3 (AF3293, Affinity Bioscience), STAT3 (10253-2-AP, Proteintech, Wuhan, China), p-STAT1 (28977-1-AP, Protientech), STAT1 (10144-2-AP, Proteintech), FXR (M022312, Abmart, Shanghai, China), and GAPDH (endogenous control, 60004-1-Ig, Proteintech) overnight at 4 °C (dilution 1:1000).

Techniques: Cell Function Assay, Transfection, Plasmid Preparation, Western Blot, Flow Cytometry, Quantitative RT-PCR