Journal: bioRxiv

Article Title: Pro- and anti-inflammatory macrophages adjust UCP2 protein levels based on their intrinsic metabolism and available metabolites

doi: 10.1101/2025.09.08.674165

Figure Lengend Snippet: (A) Scheme of differentiation and polarization of bone marrow-derived macrophages into pro- and anti-inflammatory phenotypes. (B) Representative WB showing STAT1 and STAT6 as well as their phosphorylated forms (pSTAT1 and pSTAT6) in LPS-MΦ and IL4-MΦ, respectively. (C) Representative WB confirming the expression of UCP2 in pro-ad anti-inflammatory macrophages. Recombinant mouse UCP2 (1 ng) and spleen, and skeletal muscle (SkM) were used as positive and negative controls for UCP2 expression, respectively. 20 µg total cell or tissue protein was loaded per lane.

Article Snippet: Antibodies against STAT1 (9172, Cell Signaling Technology, Inc., Massachusetts, USA), p-STAT1 (9167, Cell Signaling Technology, Inc., Massachusetts, USA), STAT6 (9162, Cell Signaling Technology, Inc., Massachusetts, USA), p-STAT6 (9361, Cell Signaling Technology, Inc., Massachusetts, USA), bcl2 (3498, Cell Signaling Technology, Inc., Massachusetts, USA), BAX (2772, Cell Signaling Technology, Inc., Massachusetts, USA), NFKBIA/IkB alpha (H-4) (sc-1643, Santa Cruz Biotechnology Inc., USA), and α-tubulin (801202, Bio Legend Inc., San Diego, USA) were used at dilutions 1:1000, and 1:10000, respectively.

Techniques: Derivative Assay, Expressing, Recombinant

Journal: The American journal of pathology

Article Title: Macrophage-Kruppel-Like Factor 6 Signaling Promotes Experimental Atherogenesis.

doi: 10.1016/j.ajpath.2025.05.014

Figure Lengend Snippet: Figure 7 KLF6 promotes proinflammatory gene expression in macrophages through IRF1. A: Lyz2cre and Klf6fl/fl:Lyz2cre mice bone marrowederived macrophages (BMDMs) were stimulated with 10 ng/mL interferon (IFN)-g for 1 hour. Total protein samples were analyzed for phosphorylated STAT1 (pSTAT1) and total STAT1 expression by Western blot analyses. The pSTAT1 densitometry analysis was performed by using ImageJ software version 1.54f. B and C: Lyz2cre and Klf6fl/fl:Lyz2cre mice BMDMs were stimulated with 10 ng/mL IFN-g or tumor necrosis factor (TNF) for 6 hours. B and C: Total RNA samples were analyzed for expression of Klf6 (B) and Irf1 (C) by quantitative RT-PCR. D: Lyz2cre and Klf6fl/fl:Lyz2cre mice BMDMs were challenged with 10 ng/mL TNF for 6 hours. Total protein samples were analyzed for IRF1 expression by Western blot analysis. IRF1 densitometry analysis was performed by using ImageJ software version 1.54f. b-Actin was used as a housekeeping gene. E and F: Aortic macrophages were obtained from Lyz2cre:Apoe/ and Klf6fl/fl:Lyz2cre:Apoe/ mice fed on a control or Western-style high-fat diet (HFD) for 20 weeks. Total RNA (n Z 5) and protein (n Z 3) samples derived from these macrophages were evaluated for expression of IRF1 mRNA and protein levels by quantitative RT-PCR and Western blot, respectively. G and H: RAW264.7 cells were transfected with an IRF-luciferase reporter construct in the presence of pCIneo-Klf6 plasmid (G) or Klf6-specific siRNA (H). These cells were stimulated with 10 ng/mL TNF for 18 hours, and cell lysates were analyzed for luciferase activity. I: RAW264.7 cells were cotransfected with a combination of Klf6-specific siRNA or Mac_N_IRF1 and stimulated with 10 ng/mL TNF for 6 hours. Total RNA from these experiments was evaluated for expression of Vcam1, Ifi44, Nlrp3, and Icam1 by quantitative RT-PCR. AeG: Data were analyzed by analysis of variance followed by Bonferroni post-testing. All values are reported as means SD (AeE and GeI). n Z 3 (A, D, G, and H); n Z 4 (B, C, and I). ***P < 0.001. NS, not significant; PBS, phosphate-buffered saline.

Article Snippet: Anti-IRF1 (8478S), phosphorylated STAT1 (9167S), STAT1 (14994S), and anti-F4/80 (70076S) antibodies were obtained from Cell Signaling (Danvers, MA).

Techniques: Gene Expression, Expressing, Western Blot, Software, Quantitative RT-PCR, Control, Derivative Assay, Transfection, Luciferase, Construct, Plasmid Preparation, Activity Assay, Saline

Journal: Frontiers in Immunology

Article Title: IRF-7 Mediates Type I IFN Responses in Endotoxin-Challenged Mice

doi: 10.3389/fimmu.2020.00640

Figure Lengend Snippet: Constitutive expression of IRF-7 in resting macrophages is sustained by constitutive IFNAR signaling and STAT1 binding to the Irf7 enhancer. (A) Real-time PCR analysis of Irf7 gene expression in resting BMDMs from MyD88, TRIF, IRF-3, IFNAR1, and STAT1 knockout mice, compared to wild-type control littermates. Irf7 expression was normalized to Gapdh , and expressed relative to the levels observed in un-stimulated wild-type control cells. Data are presented as mean ± SEM of at least three independent experiments. One-way ANOVA was used to calculate statistical differences (* p < 0.05). (B,C) Western immunoblot analysis of total IRF-7 (B) and total IRF-3 (C) protein expression in whole cell lysates of resting BMDMs from MyD88, TRIF, IFNAR1, and STAT1 knockout mice, compared to wild-type control littermates. Data are representative of at least two independent experiments. (D) ChIP analysis of STAT1 binding at the IRF-7 enhancer in resting BMDMs from STAT1 and IFNAR1 knockout mice compared to wild-type control littermates. ChIP-enriched DNA was normalized to input DNA and expressed relative to the levels observed in STAT1 ChIP in un-stimulated wild-type control cells. Data shown are presented as mean ± SEM of at least three independent experiments. One-way ANOVA was used to calculate statistical differences (* p < 0.05).

Article Snippet: STAT1-deficient (Stat1 tm1Rds ) mice were from Taconic Biosciences, Inc. (Hudson, NY, USA).

Techniques: Expressing, Binding Assay, Real-time Polymerase Chain Reaction, Gene Expression, Knock-Out, Control, Western Blot

Journal: Frontiers in Immunology

Article Title: IRF-7 Mediates Type I IFN Responses in Endotoxin-Challenged Mice

doi: 10.3389/fimmu.2020.00640

Figure Lengend Snippet: Autocrine/paracrine IFNAR1-STAT1 signaling is required for IFN-β gene and protein expression in LPS-challenged macrophages. Real-time PCR and ELISA analysis of IFN-β gene and protein expression of BMDMs from IFNAR1 knockout mice (A,B) , and STAT1 knockout mice (C,D) , compared to wild-type control littermates, stimulated or not with 100 ng/ml LPS for 0–12 h. Ifnb1 expression was normalized to Gapdh and expressed relative to the levels observed in un-stimulated wild-type control cells. Data are presented as mean ± SD of duplicate determinations from one representative of at least three independent experiments (N.D.: not detected).

Article Snippet: STAT1-deficient (Stat1 tm1Rds ) mice were from Taconic Biosciences, Inc. (Hudson, NY, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Knock-Out, Control

Journal: Frontiers in Immunology

Article Title: IRF-7 Mediates Type I IFN Responses in Endotoxin-Challenged Mice

doi: 10.3389/fimmu.2020.00640

Figure Lengend Snippet: TBK1 is required for the activation of both IRF-7 and IRF-3 downstream of TRIF for optimal IFN-β expression in LPS-challenged macrophages. (A,B) Real-time PCR and ELISA analysis of IFN-β gene and protein expression of BMDMs from IRF-3, IRF-7, and TRIF single knockout mice, and IRF-3-IRF-7 double knockout mice, compared to wild-type control littermates, stimulated or not with 100 ng/ml LPS for the indicated times. Ifnb1 expression was normalized to Gapdh and expressed relative to the levels observed in un-stimulated wild-type control cells. Data are presented as mean ± SD of duplicate determinations from one representative of at least two independent experiments (N.D.: not detected). (C) Western immunoblot analysis of phospho-STAT1 and total STAT1 protein expression in whole cell lysates of BMDMs from IRF-3, IRF-7, and TRIF single knockout mice, and IRF-3-IRF-7 double knockout mice, compared to wild-type control littermates, stimulated or not with 100 ng/ml LPS for 0–6 h. Data are representative of at least two independent experiments. (D) Real-time PCR analysis of IFN-β gene expression in BMDMs from IRF-3 single knockout mice, IRF-7 single knockout mice, and IRF-3-IRF-7 double knockout mice, compared to wild-type control littermates, pre-treated or not with 2 μM BX795 (TBK1 inhibitor) for 1 h, and then stimulated or not with 100 ng/ml LPS for 0–2 h. Ifnb1 expression was normalized to Gapdh and expressed relative to the levels observed in un-treated and un-stimulated wild-type control cells. Data are presented as mean ± SD of duplicate determinations from one representative of at least two independent experiments.

Article Snippet: STAT1-deficient (Stat1 tm1Rds ) mice were from Taconic Biosciences, Inc. (Hudson, NY, USA).

Techniques: Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Knock-Out, Double Knockout, Control, Western Blot, Gene Expression

Journal: iScience

Article Title: AF6 regulates intestinal IgA via crosstalk between intestinal epithelial cells and immune cells in inflammatory bowel disease

doi: 10.1016/j.isci.2025.112658

Figure Lengend Snippet: AF6 regulates the expression of MHC II by modulating the expression of STAT1 in intestinal epithelial cells (IECs) (A) The protein levels of components of the IFN-γ-related signaling pathway were detected by immunoblotting of colon tissues from Af6 f/f and Af6 ΔIEC mice maintained on 2.5% DSS for 7 days. (B) qPCR was used to assess STAT1 transcript levels in colon tissues from Af6 f/f and Af6 ΔIEC mice maintained on 2.5% DSS for 7 days. (C and D) (C) The mRNA levels of STAT1 and its downstream target genes (as assessed by qPCR) and (D) the protein levels of STAT1 and proteins encoded by its downstream target genes (as assessed by immunoblotting) were determined in organoids generated from colon tissues of Af6 f/f and Af6 ΔIEC mice, as measured before and after IFN-γ treatment. (E) Co-immunoprecipitation (co-IP) was used to detect endogenous interactions between AF6 and IRF1 in IECs. (F) 293T cells were co-transfected with constructs encoding hemagglutinin-tagged AF6 (HA-AF6) and Flag peptide-tagged IRF1 (Flag-IRF1); their interaction domains were detected by co-IP. (G) Immunoblotting was used to assess the expression of IRF1 and proteins encoded by its downstream genes in HT29 cells with or without IFN-γ exposure. (H) HT29 cells were transfected with constructs encoding AF6 with no or 3 nuclear localization signaling (NLS) domains (ΔNLS-AF6 and 3×NLS-AF6, respectively), and the expression and localization of IRF1 and proteins encoded by its downstream genes were assessed by immunoblotting following the separation of the nuclear and cytoplasmic fractions. Data are expressed as mean ± SEM. Pairwise comparisons between groups were conducted using two-tailed non-paired Student’s t tests. p -values were shown in the panel, and p < 0.05 indicates a significant difference.

Article Snippet: Phospho-Stat1 (Tyr701) (58D6) Rabbit mAb , Cell Signaling Technology , Cat#9167.

Techniques: Expressing, Western Blot, Generated, Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Construct, Two Tailed Test

Journal: bioRxiv

Article Title: ISG15 orchestrates dynamic crosstalk between mitochondrial fat oxidation and type 1 interferon in myeloid cells

doi: 10.64898/2026.01.19.700051

Figure Lengend Snippet: (A ) Strategy for CRISPR Cas9 mediated genetic depletion of Cpt1a in primary BMDMs. (B) Seahorse analysis of OCR in Cpt1a KO and control BMDMs on DMXAA stimulation on sequential treatment with oligomycin, FCCP and Rotenone/Antimycin A (n=8 per group). (C-D) Histograms of basal OCR (C) and maximal respiration (D) of seahorse analysis in B . (E) Histograms showing IFN-β cytokine levels in Cpt1a KO and control BMDMs (n=4 per group) on treatment with DMXAA for indicated time points (0-6h). Results measured were normalized to the cell counts in each corresponding wells using two-way ANOVA followed by Tukey’s multiple comparisons test. (F) Representative immunoblots of pSTAT1, STAT1, p-STAT2, STAT2, ISG15, p-IRF3, IRF3 and vinculin in DMXAA treated control and Cpt1a KO BMDMs upon treatment with DMXAA for indicated time points (0-6h) (n=3 per group). (G) Representative blots of Ac-His3 K9/K14, total His3 and vinculin in control and Cpt1a KO BMDMs upon treatment with DMXAA for indicated time points (0-6h) (n=3 per group). (H) Schematic of proposed epigenetic control whereby type 1 IFN in a feed forward manner increases further interferon production in response by increaing myeloid cell mitochondrial FAO to generate Acetyl-CoA and histone acetylation. Asterisks represent - * p < 0.05; ** p < 0.01; *** p < 0.001. n.s. - not significant.

Article Snippet: The following antibodies were used: STAT1, phospho-STAT1, STAT2, phospho-IRF3, ISG15, acetyl-Histone H3(Lys9/Lys14), Histone H3 (Cell Signaling Technology); phospho-STAT2 (EMD Millipore); IRF3 (Abcam); Cpt1a, HADHB (proteintech); HADHA (Invitrogen); ACAT1 and vinculin (Sigma-Aldrich).

Techniques: CRISPR, Control, Western Blot

Journal: bioRxiv

Article Title: ISG15 orchestrates dynamic crosstalk between mitochondrial fat oxidation and type 1 interferon in myeloid cells

doi: 10.64898/2026.01.19.700051

Figure Lengend Snippet: (A ) Schematic of the murine infection study design. LCMV infected C57bl/6 mice were sacrificed at indicated time points (0,1,3,5 and 8 days) post infection and splenocytes were isolated (n=5 mice per group/day). CD11b+ myeloid cells were separated from the splenocytes and used for this study. (B-G) Histograms showing quantitative RT-PCR analysis of type 1 IFN response genes Ifnb (B) , Stat1 (C) , Stat2 (D) , Isg15 (E) and mitochondrial FAO enzymes Acat1 (F) and Cpt1a (G) in CD11b+ myeloid cells isolated from the splenocytes of infected mice. Data were normalized to 18 S rRNA and represented as means ± SEM. Splenocytes from infected mice were subjected to treatment with 5µM FaO Blue followed by staining with PE conjugated CD11b+ antibody. (H) Representative data of flow cytometry based measurement of fluorescence intensity of FaO Blue in CD11b+ splenocytes from infected mice at indicated time points (J) Representation of flow cytometric geometric mean fluorescence intensity of FaO Blue in infected mice at different timepoints. (K) Representative microscopic images of CD11b+ myeloid cells, isolated from the splenocytes of infected mice at different timepoints, incubated with FaO Blue. (I) Histograms of fluorescence intensity of FaO Blue from microscopic images in I . Data represented as means ± SEM. Two-way ANOVA followed by Tukey’s multiple comparisons test. Asterisks represent * p < 0.05; ** p < 0.01; *** p < 0.001. n.s. - not significant.

Article Snippet: The following antibodies were used: STAT1, phospho-STAT1, STAT2, phospho-IRF3, ISG15, acetyl-Histone H3(Lys9/Lys14), Histone H3 (Cell Signaling Technology); phospho-STAT2 (EMD Millipore); IRF3 (Abcam); Cpt1a, HADHB (proteintech); HADHA (Invitrogen); ACAT1 and vinculin (Sigma-Aldrich).

Techniques: Infection, Isolation, Quantitative RT-PCR, Staining, Flow Cytometry, Fluorescence, Incubation

Journal: bioRxiv

Article Title: ISG15 orchestrates dynamic crosstalk between mitochondrial fat oxidation and type 1 interferon in myeloid cells

doi: 10.64898/2026.01.19.700051

Figure Lengend Snippet: (A ) Representative immunoblots of p-STAT1, STAT1, p-STAT2, STAT2, p-IRF3, IRF3 and vinculin from control and Isg15 KO BMDMs stimulated with DMXAA for different timepoints (0-12h).(n=5) (B) Volcano plot representation of differentially expressed genes in IFN-α treated PBMCs from ISG15 deficient patients compared to IFN-α treated PBMCs from healthy volunteers (GSE60359). x-axis represents log2 fold change in gene expression, and y-axis represents the −log10 adjusted p-value. Each point corresponds to a gene. Genes with large fold changes and high statistical significance appear toward the top left and top right of the plot, highlighting significantly downregulated and upregulated genes, respectively. Genes associated with type 1 IFN response has been highlighted as yellow dots. (C-E) Dot plot representation of scRNA analysis to understand the expression pattern of FAO associated genes in different immune cells from dermal tissues of healthy volunteers (C) , DLE (D) and SLE (E) patients. Dot size represents the percentage of immune cells expressing each gene and dot color represents the average expression level of indicated genes. (F) Schematic of the clinical validation study to determine FAO in CD14+ myeloid cells from PBMCs of control vs. SLE patients. PBMCs isolated from blood of 9 SLE patients and age matched healthy volunteers were incubated with 5 µM FaO Blue for 1h followed by staining with APC conjugated anti CD14 antibody. The CD14+ populations were gated and analyzed for fluorescent intensity of FaO blue by flow cytometry. (G) Representative data of flow cytometry based measurement of FaO Blue intensity in CD14+ gated PBMCs from SLE patient and respective age matched healthy volunteer. (H) Dot plot of paired geometric mean fluorescent intensity of FaO Blue in CD14+ gated PBMCs from SLE patient and respective age matched healthy volunteer (n=9). (I-M) Quantitative RT-PCR analysis of ISG15 (I) , CPT1A (J) , ACAT1 (K) , HADHA (L) and HADHB (M) in CD14+ myeloid cells isolated from PBMCs of SLE patients and respective age matched healthy volunteers. Data were normalized to 18 S rRNA. Paired t-test. (N) The heatmap shows expression profiling by array data from the GSE60359 dataset, comparing healthy controls and ISG15-deficient patients. Rows represent the genes of key FAO enzymes CRAT , ECHS1 , HADHA , ACAT1 , CPT1A , HADHB , PPARG , and PPARA , while columns represent six individual samples. Expression values were normalized using quantile normalization and scaled to z-scores. Colors indicate relative gene expression levels, as shown in the color bar. Asterisks represent - * p < 0.05; ** p < 0.01; *** p < 0.001. n.s. - not significant.

Article Snippet: The following antibodies were used: STAT1, phospho-STAT1, STAT2, phospho-IRF3, ISG15, acetyl-Histone H3(Lys9/Lys14), Histone H3 (Cell Signaling Technology); phospho-STAT2 (EMD Millipore); IRF3 (Abcam); Cpt1a, HADHB (proteintech); HADHA (Invitrogen); ACAT1 and vinculin (Sigma-Aldrich).

Techniques: Western Blot, Control, Gene Expression, Expressing, Biomarker Discovery, Isolation, Incubation, Staining, Flow Cytometry, Quantitative RT-PCR