Journal: bioRxiv

Article Title: Human iPSC derived alveolar macrophages reveal macrophage subtype specific functions of itaconate in M. tuberculosis host defense

doi: 10.1101/2025.07.23.664455

Figure Lengend Snippet: (A) Mtb colony forming units (CFUs) quantified from wild type (green symbols) and Irg1 -/-(gold symbols) MCDM (shaded bars) and AM-L macrophages (clear bars), infected at an MOI of 3, by day of infection (B) Relative concentration of CXCL1 or CCL4 produced by WT or Irg1 -/- MCDM (shaded bars) and AM-L (clear bars) macrophages on day 3 of a Mtb infection. (C) Relative concentration of IL1-β and IL-1RA produced by WT and Irg1 -/- MCDM and AM-L macrophages on day 3 of a Mtb infection. (D) Itaconate negatively regulates the Type I interferon response to Mtb in MCDM but not AM-L macrophages. Heatmap depicting z-score of hallmark Type I interferon gene expression in MCDM and AM-L macrophages in the presence or absence of Mtb. Partitioning is based on k-means clustering. (E) Hyperinduction of the Type I IFN pathway in itaconate deficient macrophages is due to phagosomal permeabilization by esx-1 . RT-qPCR quantifying Trim5, Isg20, and Cxcl11 transcripts, normalized to GAPDH, in MCDM macrophages in response to infection by wildtype Mtb or MtbΔ rv3877 (lacking the esx-1 secretion system) for four hours. ** P value <0.01 **** P value <0.0001 by two-way ANOVA. Error bars are SD.

Article Snippet: This assay used TRIM5 (ThermoFisher, Hs01552558_m1) conjugated to FAM, ISG20 (ThermoFisher, Hs00158122_m1) conjugated to FAM, or CXCL11 (ThermoFisher, Hs00171138_m1) conjugated to FAM.

Techniques: Infection, Concentration Assay, Produced, Gene Expression, Quantitative RT-PCR

Journal: BioFactors (Oxford, England)

Article Title: Pancreatic β-cell Production of CXCR3 Ligands Precedes Diabetes Onset

doi: 10.1002/biof.1304

Figure Lengend Snippet: A. Cxcl9, Cxcl10 and Cxcl11 transcript levels were measured in islets isolated from 10 week old male (n = 4) and female NOD mice (n = 5). Data are expressed relative to age-matched BALB/c control mice (n = 7). **p<0.01, *p<0.05. B–F. Islets from Wistar rats (B, C; n = 4 per group) or human islets (D–F; n =3 per group) were untreated (NT) or stimulated with 10 ng/mL IL-1β, 100 U/mL IFN-γ or both cytokines for 3 h. B–F. Relative mRNA abundance of CXCL9 (B, D), CXCL10 (E) and CXCL11 (C, F) was determined by RT-PCR. ***p<0.001 vs. NT, **p<0.01 vs. NT, *p<0.05 vs. NT.

Article Snippet: Serum levels of CXCL9, CXCL10, and CXCL11 were detected using the mouse CXCL9 Quantikine ELISA kit (Cat # MCX900), CXCL10 Quantikine ELISA kit (Cat # MCX100) and the mouse CXCL11 DuoSet ELISA kit (Cat # DY572), all from R&D Systems (Minneapolis, MN) using the protocol provided by the manufacturer.

Techniques: Isolation, Control, Reverse Transcription Polymerase Chain Reaction

Journal: BioFactors (Oxford, England)

Article Title: Pancreatic β-cell Production of CXCR3 Ligands Precedes Diabetes Onset

doi: 10.1002/biof.1304

Figure Lengend Snippet: A–C. 832/13 rat insulinoma cells were stimulated with 1 ng/mL IL-1β or IL-1β plus 100 U/mL IFN-γ for the indicated times (NT; no treatment). D–F. 832/13 cells were pretreated for 1 h with either DMSO or 0.5 μg/mL Cycloheximide (CHX). Cells were subsequently exposed to IL-1β (1 ng/mL) or the combination of IL-1β and IFN-γ (100 U/mL) for 2 h. Cellular mRNA levels of Cxcl9 (A, D), Cxcl10 (B, E) and Cxcl11 (C, F) were detected by RT-PCR. n.s. = not significant vs respective treatment in DMSO control group. Data are shown as means ± SEM from three independent experiments.

Article Snippet: Serum levels of CXCL9, CXCL10, and CXCL11 were detected using the mouse CXCL9 Quantikine ELISA kit (Cat # MCX900), CXCL10 Quantikine ELISA kit (Cat # MCX100) and the mouse CXCL11 DuoSet ELISA kit (Cat # DY572), all from R&D Systems (Minneapolis, MN) using the protocol provided by the manufacturer.

Techniques: Reverse Transcription Polymerase Chain Reaction, Control

Journal: BioFactors (Oxford, England)

Article Title: Pancreatic β-cell Production of CXCR3 Ligands Precedes Diabetes Onset

doi: 10.1002/biof.1304

Figure Lengend Snippet: A. 832/13 cells were exposed to 100 U/mL IFN-γ for the indicated times. PO4-STAT1Y701 and total STAT1 protein abundance were determined by immunoblotting. B, C. 832/13 cells were pre-treated for 1 h with increasing concentrations of JAKi (1 nM, 10 nM, 100 nM), followed by a 3 h stimulation with IL-1β alone (1 ng/mL) or IL-1β plus 100 U/mL IFN-γ. ***p<0.001 vs. DMSO (black bar), *p<0.05 vs. DMSO (black bar). D, E. 832/13 cells were transfected with two siRNA duplexes targeting STAT1 using a scrambled siRNA sequence duplex as a control. 48 h post- transfection cells were cultured for 3 h with 1 ng/ml IL-1β or IL-1β plus 100 U/ml IFN-γ. ***p<0.001 vs. siScramble (black bar), *p<0.05 vs. siScramble (black bar). Cxcl9 (B, D) and Cxcl11 (C, E) mRNA levels were quantified. Data are represented as means ± SEM from three independent experiments. The immunoblot in A was repeated on two separate occasions.

Article Snippet: Serum levels of CXCL9, CXCL10, and CXCL11 were detected using the mouse CXCL9 Quantikine ELISA kit (Cat # MCX900), CXCL10 Quantikine ELISA kit (Cat # MCX100) and the mouse CXCL11 DuoSet ELISA kit (Cat # DY572), all from R&D Systems (Minneapolis, MN) using the protocol provided by the manufacturer.

Techniques: Quantitative Proteomics, Western Blot, Transfection, Sequencing, Control, Cell Culture

Journal: BioFactors (Oxford, England)

Article Title: Pancreatic β-cell Production of CXCR3 Ligands Precedes Diabetes Onset

doi: 10.1002/biof.1304

Figure Lengend Snippet: A. 832/13 cells were transduced with adenoviruses encoding either βGAL, wild-type STAT1 (WT), STAT1Y701F, STAT1S727A, STAT1Y701F/S727A (DM; double mutant) or STAT1S727T. STAT1 abundance was determined by immunoblotting. B, C. 832/13 cells were transduced with the adenoviruses indicated in (A); 24 h post-transduction cells were stimulated for 3 h with either IL-1β (1 ng/mL) alone or IL-1β plus IFN-γ (100 U/mL). *p<0.05, #p<0.1. D, E. Rat islets were transduced with the indicated adenoviruses. 24 h post-transduction cells were stimulated with both IL-1β (10 ng/mL) and IFN-γ (100 U/mL) for 3 h. ***p<0.001, **p<0.01. Relative mRNA abundance of Cxcl9 (B, D) and Cxcl11 (C, E) was determined by RT-PCR. Date are expressed as means ± SEM from 3 (B, C) or 2 (D, E) individual experiments. The immunoblot in A was repeated on two individual occasions.

Article Snippet: Serum levels of CXCL9, CXCL10, and CXCL11 were detected using the mouse CXCL9 Quantikine ELISA kit (Cat # MCX900), CXCL10 Quantikine ELISA kit (Cat # MCX100) and the mouse CXCL11 DuoSet ELISA kit (Cat # DY572), all from R&D Systems (Minneapolis, MN) using the protocol provided by the manufacturer.

Techniques: Transduction, Mutagenesis, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: BioFactors (Oxford, England)

Article Title: Pancreatic β-cell Production of CXCR3 Ligands Precedes Diabetes Onset

doi: 10.1002/biof.1304

Figure Lengend Snippet: Schematic representation of distal and proximal GAS sites in the Cxcl9 and Cxcl11 promoters are shown (left panels). Arrows are the diagrammatic depiction of PCR amplicons. A–D. 832/13 cells were stimulated with 100 U/mL IFN-γ for either 20 mins (middle panels) or a time course (right panels). ChIP assays were performed to determine relative occupancy of total STAT1 (middle panels) and PO4-STAT1Y701 (right panels) on the Cxcl9 proximal (A) and distal promoter (B), and on the Cxcl11 proximal (C) and distal (D) promoter. ***p<0.001 vs. NT, **p<0.01 vs. NT, *p<0.05 vs. NT. Data are expressed as means ± SEM from 3–4 individual experiments.

Article Snippet: Serum levels of CXCL9, CXCL10, and CXCL11 were detected using the mouse CXCL9 Quantikine ELISA kit (Cat # MCX900), CXCL10 Quantikine ELISA kit (Cat # MCX100) and the mouse CXCL11 DuoSet ELISA kit (Cat # DY572), all from R&D Systems (Minneapolis, MN) using the protocol provided by the manufacturer.

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Patient Derived Colonoids as Drug Testing Platforms–Critical Importance of Oxygen Concentration

doi: 10.3389/fphar.2021.679741

Figure Lengend Snippet: Chemokines released by colonoids at 20 or 2% O 2 . Conditioned medium from colonoids at 20 or 2% O 2 , followed by 24h treatment with TNF, IL17 or TNF/IL17, and untreated colonoids were analyzed for chemokine concentration (A) Mean protein concentration ( p g . ml −1 ) of chemokines in conditioned medium from colonoids ( n = 3 independent assays) analyzed by Human Chemokine Panel featuring 40 magnetic bead-based immunoassays. The 18 heatmaps show expression of significantly regulated proteins and are grouped according to 1) Highest protein expression by TNF/IL17 treatment at 20% O 2 . 2) Highest protein expression by TNF/IL17 treatment at 2% O 2 . 3) Highest protein expression by TNF treatment at 20% O 2 , and 4) Highest protein expression by TNF or TNF/IL17 treatment, similar at 20 and 2% O 2 (B) CCL20, CXCL1, CXCL2, CXCL5, CXCL8, CXCL10 and CXCL11 protein concentrations ( p g.ml −1 ) in conditioned medium from minimum six assays measured by ELISA. Left panels: paired data at 2 or 20% O 2 across all treatments for the selected chemokines, analyzed with Wilcoxon matched-pairs signed rank test. Significance level is indicated as p values or not significant (ns). Right panels: concentrations for each chemokine in 2% (blue) or 20% (red) O 2 plotted as individual values with mean and SD. Statistical analysis were performed on log2 transformed data . Alterations across different treatments within each oxygen concentrations were analyzed with one-way ANOVA followed by Tukey’s multiple comparisons or Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Alterations across different oxygen concentrations within same treatment were analyzed by two-way ANOVA followed by Holm-Šídák multiple comparisons test. * p < 0.05; red and blue asterisks show comparisons across different treatments within 20 and 2% O 2 conditions, respectively. Black asterisks indicate significant comparisons between 2 and 20% oxygen concentrations within a specific treatment.

Article Snippet: ELISA kits for human CXCL1 (DY275-05), CXCL2 (DY276-05), CXCL5 (DY254-05), CXCL8 (DY208), CXCL10 (DY266-05), CXCL11 (DY672) and CCL20 (DY360), all from R&D Systems (Abington, United Kingdom), were used according to manufacturer’s protocols.

Techniques: Concentration Assay, Protein Concentration, Expressing, Enzyme-linked Immunosorbent Assay, Transformation Assay