Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Immunophenotyping panel for multiplexed tissue imaging of cancer.

Article Snippet: CD8 , REA734 , 50 , 130-110-677 , FITC , Miltenyi Biotec.

Techniques: Imaging

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

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