Journal: iScience

Article Title: TRAIL-dependent apoptosis of peritoneal mesothelial cells by NK cells promotes ovarian cancer invasion

doi: 10.1016/j.isci.2023.108401

Figure Lengend Snippet: Ascites blocks NK cell degranulation but not TRAIL-dependent HPMC apoptosis (A and B) TAL were pre-cultured in RPMI/5% AB media or 100% ascites pool (+/− α-CD3 Ab stimulation) prior to co-culture with HPMC. (A) Apoptosis of HPMC is shown as percentage of annexin V+ cells after gating on CD45 − cells (n = 7 patients). (B) Degranulating NK cells in response to HPMC were measured via flow cytometry and depicted as percentage of CD335+/CD107a+ cells (n = 6 patients). (C and D) TAL were pretreated with α-TRAIL blocking Ab prior to HPMC co-culture. An irrelevant mouse IgG was included as control. Experiments were conducted with TAL cultured in RPMI/5% AB media (n = 11 patients) (C) and 100% ascites pool (n = 5 patients) (D). The amount of Annexin V+ HPMC was determined as described previously. (E) Schematic representation of co-culture experiments applying T cell CM for NK cell activation. CM were collected from CD3 + , CD3+/CD4+, and CD3+/CD8+ T cell subsets cultured in media +/− α-CD3 Ab stimulation. Purified NK cells were then stimulated with T cell CM prior to HPMC co-cultures. (F) The amount of apoptotic HPMC induced by NK cells activated with CM of CD3 + T cells was compared with CM of CD3+/CD4+ and CD3+/CD8+ T cells (n = 4 matched pairs of different patients). (G) Analysis of TRAIL signaling was performed by applying an α-TRAIL blocking Ab to NK cells stimulated with CD3 + T cell CM (α-CD3 Ab activated) prior to the co-culture with HPMC (n = 5 patients). An irrelevant mouse IgG was included as control. The mean is shown by horizontal bars or boxes; vertical error bars represent the standard deviation in A–D, F, and G. ∗ FDR < 0.05; ∗∗ FDR < 0.01; ∗∗∗ FDR < 0.001; determined by paired t test and Benjamini-Hochberg adjustment (ns, not significant).

Article Snippet: To further divide the CD3 + T cell fraction into CD3+/CD4+ T helper cells and CD3+/CD8+ cytotoxic T-cells, the preselected CD14 − lymphocytes were stained with APC-labelled anti-human CD8 antibody (Miltenyi Biotec), incubated with anti-APC microbeads and isolated via MACS.

Techniques: Cell Culture, Co-Culture Assay, Flow Cytometry, Blocking Assay, Control, Activation Assay, Purification, Standard Deviation

Journal: iScience

Article Title: TRAIL-dependent apoptosis of peritoneal mesothelial cells by NK cells promotes ovarian cancer invasion

doi: 10.1016/j.isci.2023.108401

Figure Lengend Snippet:

Article Snippet: To further divide the CD3 + T cell fraction into CD3+/CD4+ T helper cells and CD3+/CD8+ cytotoxic T-cells, the preselected CD14 − lymphocytes were stained with APC-labelled anti-human CD8 antibody (Miltenyi Biotec), incubated with anti-APC microbeads and isolated via MACS.

Techniques: Control, Recombinant, Polymer, Software

Journal: Frontiers in Pharmacology

Article Title: Mycobacterium tuberculosis infection drives osteoclast overactivation via α2,3-Sialylation to promote pathological bone destruction

doi: 10.3389/fphar.2026.1738896

Figure Lengend Snippet: α2,3-sialylation is required for BCG-induced osteoclast differentiation and activity (A) Comparison of differentially expressed genes (DEGs) between BCG-infected osteoclasts (RB) and uninfected controls (R). (n = 3 biological replicates per group, differential expression was defined as |FoldChange| > 2 and padj <0.05). (B) Volcano plot of DEGs highlighting genes related to sialic acid biosynthesis. (C) KEGG pathway enrichment analysis of upregulated DEGs in RB cells. (D,E) Immunofluorescence staining of α2,3-SA in mouse calvarial sections using MAL II lectin, with quantification of α2,3-SA fluorescence intensity (n = 5). Intensity density was normalized to the PBS group mean. (F) In vitro osteoclasts subjected to MAL II lectin staining and TRAP staining, with or without sialidase treatment to enzymatically remove α2,3-SA. (G) Quantification of α2,3-SA fluorescence intensity in cultured osteoclasts (n = 5). Intensity density was measured in cellular ROIs after background subtraction and normalized to the RANKL group mean. (H) Quantification of TRAP + multinucleated cells (≥3 nuclei) per field (randomly selected fields, fixed magnification) in vitro (n = 5). (I) mRNA expression levels of osteoclast differentiation markers ( Fos, Mmp9, Nfatc1, and Ocstamp ) in osteoclasts (n = 3). Data are presented as mean ± SD. Statistical significance was determined by two-tailed unpaired Student’s t-test for two-group comparisons (E) and one-way ANOVA followed by Tukey’s post hoc test for three-group comparisons (G–I) .

Article Snippet: Biotin MAL-II , Vector laboratories , Cat# B-1265-1.

Techniques: Activity Assay, Comparison, Infection, Quantitative Proteomics, Immunofluorescence, Staining, Fluorescence, In Vitro, Cell Culture, Expressing, Two Tailed Test

Journal: Journal of Clinical Investigation

Article Title: Leukotriene B4 amplifies NF-κB activation in mouse macrophages by reducing SOCS1 inhibition of MyD88 expression

doi: 10.1172/jci43302

Figure Lengend Snippet: Figure 3 LTB4 is required for MyD88 expression in phagocytes. (A) MyD88, TIRAP, TRIF, and TIRP protein expression in WT macrophages and in 5-LO–/– and BLT1–/– macrophages treated for 24 hours with or without LTB4. (B) Densitometry of MyD88 protein in 5-LO–/– and BLT1–/– mac- rophages. (C) Myd88 mRNA expression in 5-LO–/– and BLT1–/– macrophages treated as in A. (B and C) Data are representative of 3 experi- ments, performed in triplicate; values are relative to control WT macrophages (dashed line). *P < 0.05 versus WT; #P < 0.001 versus untreated 5-LO–/–. (D) MyD88 protein expression in resident peritoneal (PM), alveolar (AM), and splenic (SM) macrophages as well as splenic DCs, T and B lymphocytes, and lung fibroblasts from WT and 5-LO–/– mice. Data are representative of 3 experiments. (E) Myd88 mRNA decay in WT and 5-LO–/– macrophages harvested after treatment with actinomycin D (2.5 mg/ml). Data are from 3 experiments in triplicate; values are rela- tive to untreated macrophages from both genotypes. (F) MyD88 protein in WT and 5-LO–/– macrophages incubated for 24 hours as indicated. Immunoblot results are from 2–3 independent experiments (relative MyD88 density shown by numbers beneath). Lanes were run on the same gel but were noncontiguous (white line). (G) Nitrite production in WT and 5-LO–/– macrophages pretreated for 24 hours with or without LTB4, followed by LPS for another 24 hours. Data are representative of 3 experiments. *P < 0.05 versus control; #P < 0.05 versus non–LTB4-treated stimulated WT; &P < 0.01 versus LPS or IL-1β alone.

Article Snippet: Protein samples were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with commercially available primary antibodies against MyD88, TRIF, TIRP, SOCS1 (all 1:500, Abcam); p65, TIRAP, phosphorylated IκBα, p50, p65 (all 1:500, Santa Cruz); Tyr701and Ser727-phosphorylated as well as total STAT1, STAT3, and JAK2 and phosphorylated JAK2 (Tyr1007/1008), JAK1 (Tyr1022/1023), Tyk2 (Tyr1052/1053), and STAT3 (Tyr705) (all 1:1,000; Cell Signaling); and β-actin (1:10,000; Sigma-Aldrich).

Techniques: Expressing, Control, Incubation, Western Blot