Journal: bioRxiv

Article Title: Activation-induced shift in nutrient preference and function-specific nutrient dependence in human neutrophils

doi: 10.1101/2023.09.25.559385

Figure Lengend Snippet: a. Schematic of glucose uptake, glycogen synthesis, and glycogenolysis. Important enzymes are listed. b. Glucose consumed from the media within 1 hour by unstimulated neutrophils or neutrophils that are treated with indicated stimuli (300μg/mL zymosan A, 100ng/mL TNFα, or 100nM PMA) or inhibitor (20μM GPi). Bars and error bars show mean ± SD from n=3-4 independent donors, as represented by individual dots. Statistical analysis was done by unpaired t-test, * indicates p<0.05. c. (Left) Representative flow cytometry result measuring the distribution of GLUT1 level on cell surface within a population of neutrophils that are untreated or treated with indicated stimuli or inhibitor for 30min. (Right) The experiment was repeated with neutrophils isolated from n=4 independent donors (indicated by different markers). Bars and error bars show mean ± SD of quantified mean GLUT1 level from each donor. Statistical analysis was performed by paired t-test. d-e. Immunoblot analysis of (d) total GLUT1 level and phosphorylated GLUT1 (Ser226), and (e) total PYGL and phosphorylated PYGL (Ser15) in neutrophils treated with indicated stimuli or inhibitor for 30min. Representative blots were shown on the left. The experiment was repeated multiple times with neutrophils isolated for different donors (indicated by individual dots), and quantified results were compiled and shown on the right. Bars and error bars show mean ± SD. Statistical analysis performed by unpaired t-test. f. Level of glycogen store in human peripheral blood neutrophils that are untreated, treated with GPi (20μM) for 2h, or stimulated with zymosan A (300μg/mL), or LPS (1μg/mL) for 2h. Bars and error bars show mean ± SD from n=4-5 independent donors, as represented by individual dots. p-value determined by paired t-test. g. Fraction of labeled glycogen after 2hr incubation of neutrophils that are untreated or treated with indicated stimuli or inhibitor in media containing U- 13 C-glucose. Bars and error bars show mean ± SD from n=3-5 independent donors, as represented by individual dots. p-values were determined by paired t-test.

Article Snippet: Primary antibodies: PYGL (abcam, ab198268), phospho(S15)-PYGL (abcam, ab227043), GLUT1 (Alomone Labs, AGT-041), P-GLUT1 (Millipore Sigma, ABN991), B-actin (Cell Signaling, no. 4967); and secondary antibodies: Goat-anti-rabbit 800 (LI-COR, no. 925-32211), Goat-anti-mouse 680 (LI-COR, no. 925-68070).

Techniques: Flow Cytometry, Isolation, Western Blot, Labeling, Incubation