Journal: Open Life Sciences

Article Title: AUDA suppresses apoptosis of HCAECs induced by TNF and serum of Kawasaki disease patients by inhibiting endoplasmic reticulum stress

doi: 10.1515/biol-2025-1254

Figure Lengend Snippet: AUDA inhibits ERS by suppressing the JAK/STAT1 pathway. (A) 45 mmol/L of STAT1 transcription enhancer (2-NP) was used to activate the JAK/STAT1 signaling pathway. (B) Cell viability was detected using CCK8 assay. *** P < 0.001 versus TNF control, &&& P < 0.001 versus TNF and AUDA-treated cells. (B) The level of LDH was detected using Elisa assay. (C) The expression of ERS marker factors was detected. The protein levels of IRE1α (D), XBP1 (E), CHOP (F), ATF6 (G), Cleaved-Caspase12 (H) and Caspase12 (I) were standardized to GAPDH. (J) Heatmap of ERS marker factors expression. *** P < 0.001.

Article Snippet: 45 mmol/L of STAT1 transcription enhancer (2-NP) (MedChemExpress, Monmouth Junction, NJ, USA) was used to activate the Janus kinase (JAK)/STAT1 signaling pathway.

Techniques: CCK-8 Assay, Control, Enzyme-linked Immunosorbent Assay, Expressing, Marker

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: 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: 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