Journal: Cell reports

Article Title: Nanoparticle-based itaconate treatment recapitulates low-cholesterol/low-fat diet-induced atherosclerotic plaque resolution

doi: 10.1016/j.celrep.2024.114911

Figure Lengend Snippet:

Article Snippet: Direct-zol RNA Miniprep Kit , Zymo Research , R2054.

Techniques: Purification, Plasmid Preparation, Produced, Recombinant, Concentration Assay, Saline, Labeling, Membrane, Enzyme-linked Immunosorbent Assay, Quantitation Assay, Phospholipid Assay, BIA-KA, RNAscope, HD Assay, Polymer, Sequencing, Expressing, Software, Microscopy

Journal: PLOS Biology

Article Title: Multiomic profiling of chronically activated CD4+ T cells identifies drivers of exhaustion and metabolic reprogramming

doi: 10.1371/journal.pbio.3002943

Figure Lengend Snippet: (A) Diagram depicting the culture model used to generate time course samples from healthy human CD4+ memory T cells. Cells were stimulated with CD3/CD28 Dynabeads for 12 days, with fresh beads plus full media change every other day (fresh media alone added on alternate days). Samples were collected on days highlighted in bold. (B) Multiomics workflow (see for details). Experiments were performed in biological triplicate, with proteomics, phosphoproteomics, and metabolomics done on same samples. (C) ELISA-based quantification of secreted cytokines (IL-2, IFN-γ, TNF-α) on days 0, 6, 10, and 12, 3 replicates per time point. (D) Cell counts were taken each time point, and average cell division compared to previous time point calculated. Cell viability remained stable throughout at ~75%–85%. (E) Flow cytometric analysis of inhibitory receptors PD-1, TIM-3, and TIGIT at resting, day 6, and day 12 time points. Data normalized to mode for accurate population comparisons to account for differences in number of cells analyzed. The data underlying can be found in the supplemental flow cytometry files uploaded to flowrepository.org . Figure created with BioRender.com .

Article Snippet: After aspiration, cells were resuspended in PBS with 1% BSA (Thermo, 15260037) and 50 mM EDTA, and T cells were isolated using untouched memory CD4+ T-cell isolation kit (Miltenyi, 130-091-893).

Techniques: Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: PLOS Biology

Article Title: Multiomic profiling of chronically activated CD4+ T cells identifies drivers of exhaustion and metabolic reprogramming

doi: 10.1371/journal.pbio.3002943

Figure Lengend Snippet: Mean fluorescence intensity of PD-1 in CD4+ T cells from both HIV+ and HIV- patients (grouped due to observing same effect in both) for CD4+ cells that are CD276+ or FLT-1+. The data underlying this figure can be found in the supplemental flow cytometry files uploaded to flowrepository.org . Figure created with BioRender.com .

Article Snippet: After aspiration, cells were resuspended in PBS with 1% BSA (Thermo, 15260037) and 50 mM EDTA, and T cells were isolated using untouched memory CD4+ T-cell isolation kit (Miltenyi, 130-091-893).

Techniques: Fluorescence, Flow Cytometry

Journal: PLOS Biology

Article Title: Multiomic profiling of chronically activated CD4+ T cells identifies drivers of exhaustion and metabolic reprogramming

doi: 10.1371/journal.pbio.3002943

Figure Lengend Snippet: (A) Causal network analysis leveraging annotated interactions (transcription factor-target, kinase-substrate, phosphatase-substrate) as a framework to evaluate protein expression changes (late exhaustion (day 12) versus resting, taking into account previous time point comparisons) to predict key nodes (regulators) impacting most changes seen across the data set. (B) Pruned version of a large subnetwork with p300 (black outline) at main hub. Orange circles mark proteins of interest mentioned in main text. (C) Depiction of experimental design of p300 inhibition studies. (D) Flow cytometric analysis of PD-1 and TIM-3 showing shifts in fluorescent intensity of cell populations in treated versus untreated (cell number normalized to mode). Cell numbers shown for each plot are 20,312 for p300 treated and 30,438 for DMSO, which corresponds to all CD4+ T cells from the sample (excluding doublets and debris, see for gating), corresponding to >95% of sample. (E) Bar chart of mean fluorescent intensity showing significant changes ( T test) in PD-1 and TIM-3 during p300 inhibition versus controls (antigen removed for 2 days, or treatment with T-cell activation inhibitor Dasatinib); *, p -value <0.05, ***, p -value <0.005. (F) Heatmap showing recruitment of cofactors (p300, class I HDACs, KAT2B) to transcription factor family DNA motifs as measured using CASCADE protein-binding microarrays. The data underlying this figure can be found in and Data (A, B), supplemental flow cytometry files uploaded to flowrepositroy.org (D), and the (E, F). Figure created with BioRender.com .

Article Snippet: After aspiration, cells were resuspended in PBS with 1% BSA (Thermo, 15260037) and 50 mM EDTA, and T cells were isolated using untouched memory CD4+ T-cell isolation kit (Miltenyi, 130-091-893).

Techniques: Expressing, Inhibition, Activation Assay, Protein Binding, Flow Cytometry

Journal: PLOS Biology

Article Title: Multiomic profiling of chronically activated CD4+ T cells identifies drivers of exhaustion and metabolic reprogramming

doi: 10.1371/journal.pbio.3002943

Figure Lengend Snippet: UMAPs of CD4+ subtypes and CD8+ T exhausted tumor lymphocytes showing gene expression of (A) exhaustion-associated genes, and (B) gene expression of proteins identified in the model presented by Lawton and colleagues. UMAPs made using Single Cell Portal, Tarhan and colleagues. The data from this figure and tool used to make it can be found at https://singlecell.broadinstitute.org/single_cell/study/SCP1039/a-single-cell-and-spatially-resolved-atlas-of-human-breast-cancers . Figure created with BioRender.com .

Article Snippet: After aspiration, cells were resuspended in PBS with 1% BSA (Thermo, 15260037) and 50 mM EDTA, and T cells were isolated using untouched memory CD4+ T-cell isolation kit (Miltenyi, 130-091-893).

Techniques: Gene Expression

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Neutrophil-macrophage crosstalk via NETs–IL-17/VEGF/S100A9 axis promotes hepatocellular carcinoma progression

doi: 10.1186/s13046-025-03618-x

Figure Lengend Snippet: The levels of NETs increase in HCC patients, especially in advanced and metastatic stages. A A heatmap illustrating the differential expression patterns of NETs-related genes in normal and HCC samples based on TCGA database analysis. B Box plots depicting the expression differences of NETs-related genes between normal and HCC samples from the TCGA database. C , D Representative images of HE (Hematoxylin and eosin) and IF staining for CD66b (C) and CitH3 (D) in tissue sections from HCs and HCC patients. The mean IOD of CD66b or CitH3 was quantified (Image Pro Plus) from three random regions per section (HC, n = 5; HCC, n = 5) and shown as fold change relative to HCs. E Representative images of IF staining for CitH3 in peripheral blood neutrophils from HC and HCC patient F Western blot analysis of CitH3 expression in peripheral blood neutrophils from HCs ( n = 8) and HCC ( n = 8) patients, with band intensities normalized to β-actin and shown as fold change relative to controls G ELISA analysis of serum MPO-DNA levels in HCs and HCC patients. H ELISA analysis of serum MPO-DNA levels in HCC patients at different TNM stages (I–IV). I ELISA analysis of serum MPO-DNA levels in HCC patients with and without metastasis. J ELISA analysis of serum MPO-DNA levels in HCC patients with intrahepatic metastasis and extrahepatic metastasis. K Western blot analysis of CitH3 expression in peripheral blood neutrophils from HCC patients with ( n = 6) and without ( n = 6) extrahepatic metastasis. Band intensities were normalized to β-actin and quantified relative to the non-metastasis group White and black scale bars: 50 μm. Data are presented as median (IQR) (G-J) and other data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001. Student’s t test (C, D, F, K), Mann–Whitney U test (B, G, I, J), Kruskal–Wallis test (H)

Article Snippet: Neutrophils were isolated from the peripheral blood of healthy volunteers and HCC patients using the Human Peripheral Blood Neutrophil Isolation Kit (P9040, Solarbio, China), according to the manufacturer’s instructions.

Techniques: Quantitative Proteomics, Expressing, Staining, Western Blot, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Neutrophil-macrophage crosstalk via NETs–IL-17/VEGF/S100A9 axis promotes hepatocellular carcinoma progression

doi: 10.1186/s13046-025-03618-x

Figure Lengend Snippet: Activation of IL-17R/NF-κB signaling by NETs-attached IL-17 is responsible for NETs-induced M2 macrophage polarization. A Schematic illustration of the transcriptome sequencing protocol for THP1-M0 under three treatment conditions. The volcano plot shows significantly DEGs for Group B vs. A, Group C vs. A, and Group C vs. B. B KEGG pathway enrichment analysis of DEGs was conducted using the DAVID tool. C FCM analysis of the proportion of CD68 + CD206 + M2 derived from THP-1-M0 following 48-hour co-culture of HuH-7 cells with NETs and (or) AMG827. Quantification of the fold change in the proportion from three independent experiments is shown in the right panel. D Western blot analysis of IL-10 and VEGF expression in cell lysates following the same treatments as in C. Densitometric values were normalized to β-actin and compared to the control group, with quantification shown in the right panel E Western blot analysis of IL-17 expression in extracted and purified NETs from peripheral blood neutrophils of HCs ( n = 8) and HCC patients ( n = 8). F ELISA analysis of IL-17 levels carried by NETs standardized to 50 ng/µL in HCs ( n = 8) and patients with HCC ( n = 8). G Western blot analysis of IL-17 expression in peripheral blood neutrophils from HCs ( n = 8) and HCC ( n = 8) patients. Densitometric values were normalized to β-actin and shown as a fold change relative to HCs. H Representative IF images of IL-17 and NE staining in peripheral blood neutrophils with (lower panel) or without PMA stimulation (upper panel) for 4 h from HCs and HCC patients. I Western blot analysis was conducted to assess the expression levels of p-65 and p-p65(S536) in THP-1-M0 following 48 h co-culture of HuH-7 cells with NETs and (or) AMG827. Densitometric values were normalized to β-actin and shown as fold changes relative to control in the right panel. J FCM was used to analyze the proportion of CD68 + CD206 + M2 derived from THP-1-M0 following 48-hour co-culture of HuH-7 cells with NETs and (or) BAY11-7082. Quantification of the fold change in the proportion from three independent experiments is shown in the right panel. K IF analysis of CD206 + M2 macrophage from the above co-culture systems of J treatment for 48 h. L ELISA analysis of secretion levels of M2d-related cytokines VEGF, IL-10 and TGF-β, in the culture supernatant from the above co-culture systems of J treatment for 48 h White scale bars: 20 μm. Data are presented as mean ± SD. Ns, not significant. * p < 0.05, ** p < 0.01, *** p < 0.001. One-way ANOVA followed by the Newman-Keuls multiple comparison test (C, D, I, J, L); Student’s t test (F, G)

Article Snippet: Neutrophils were isolated from the peripheral blood of healthy volunteers and HCC patients using the Human Peripheral Blood Neutrophil Isolation Kit (P9040, Solarbio, China), according to the manufacturer’s instructions.

Techniques: Activation Assay, Sequencing, Derivative Assay, Co-Culture Assay, Western Blot, Expressing, Control, Purification, Enzyme-linked Immunosorbent Assay, Staining, Comparison