Journal: JCI Insight
Article Title: The shifting lipidomic landscape of blood monocytes and neutrophils during pneumonia
doi: 10.1172/jci.insight.164400
Figure Lengend Snippet: ( A ) Box-and-whisker plots depicting cytokine release of monocytes (500,000 cells per well, 10 healthy donors) preincubated for 1 hour with either myriocin or PF543, and subsequently stimulated for 24 hours with lipopolysaccharide (LPS; 100 ng/mL). The y axis depicts concentration in pg/mL (±SEM, vertical bars). The colors indicate the different conditions. Significance was determined by a paired Wilcoxon’s rank-sum test. A P value below 0.05 was considered significant. * P < 0.05, ** P < 0.01. ( B ) Identical to panel A , but here for neutrophils (500,000 cells per well, 8 healthy donors), stimulated for 2 hours with LPS. ( C ) Box-and-whisker plots showing levels (in pmol/mL) of ceramide species in monocytes incubated with either vehicle or myriocin, measured by targeted MS ( n = 4 per condition). Cer, ceramide; dh-Cer, dihydroceramide; glcCer, glucosylceramide; lacCer, lactosylceramide; Gb3, globotriaosylceramide. ( D ) Box-and-whisker plots showing levels (in pmol/mL) of S1P in monocytes and neutrophils incubated with either vehicle or PF543, measured by targeted MS ( n = 4 per condition). S1P, sphingosine-1-phosphate. ( E ) Box-and-whisker plots showing intensity levels of intracellular phosphorylation markers p-ERK-171Yb (pERK), p-NF-κB-Er166 (p-NF-κB), and p-p38-Gd156 (pp38) in monocytes and neutrophils after LPS stimulation and treatment with either myriocin or PF543, measured by cytometry by time of flight (CyTOF). The y axis indicates the arsinh-normalized medium intensity levels. There were no significant differences ( n =4 per condition). ( F ) Box-and-whisker plots showing mRNA levels for key cytokines in monocytes treated with vehicle, myriocin, or PF543, measured by qRT-PCR. There were no significant differences ( n = 4 per condition).
Article Snippet: Following this, cells were washed and stained once again for intracellular phosphorylation markers pERK-171Yb, pNfkB-Er166, and pp38-Gd156 (catalog 3171010A, 3166026D, and 3156002A, respectively; Standard Biotools).
Techniques: Whisker Assay, Concentration Assay, Incubation, Phospho-proteomics, Cytometry, Quantitative RT-PCR
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