Fig 2: Metabolic outputs for extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of peripheral blood mononuclear cells in 22- and 96-week-old laying hens inoculated with Staphylococcus aureus by various routes. Outputs were collected during the Cell Energy Phenotype Test conducted (A,B) prior to inoculation and (C,D) 6 h, (E,F) 24 h, (G,H) 3 d, and (I,J) 7 d post-inoculation provided by the Seahorse XFe24 analyzer (Agilent, Santa Clara, CA). The dashed line separates measurements taken before (baseline) and after cells were stressed with simultaneous injection of FCCP and oligomycin to induce compensation by glycolytic (ECAR) or oxidative (OCR) pathways. Each data point represents the mean measurement taken from 3 hens/treatment ± SEM.

Fig 3: CISD3 depletion presents a metabolic reprogramming toward glutaminolysis.A Cell viability detection in control and CISD3-silenced cells at the presence or absence of indicated chemicals (7.5 µM Erastin, 10 µM C968, and 1 mM GPNA). B Fuel dependency assay for glucose and glutamine in control and CISD3-silenced cells. C After the glutamine oxidation pathway was inhibited by C968, the oxygen consumption (OCR) of basal and max respiration was monitored by Agilent Seahorse XFe24 Analyzer. The inhibition rate of OCR by C968 in control and CISD3-silenced cells was calculated. D, E Cell viability and cellular lipid peroxides detection in the control and CISD3-silenced cells at the presence or absence of indicated chemicals; (7.5 µM Erastin, 10 µM Rotenone (Rot), 2 mM DBM, 2.5 µM Antimycin A (Anti A), 15 mM NaN3, 5 µM CCCP). (F-I) Cell viability, lipid peroxides, cellular ROS, and mitochondrial ROS in control and CISD3-silenced cells were measured under the treatment of 7.5 µM erastin, at the presence or absence of 0.5 µM MitoQ or 0.2 µM SKQ1. The values are presented as means ± SD from three independent experiments. *P < 0.05, **P < 0.01 versus control or between different groups.

Fig 4: Prolonged stimulation through activating receptors alters the glycolytic and oxidative metabolism of NK cells.(A) NK cells harvested from isotype-coated and exhaustion plates (day 7) were immobilized on tissue culture plates and subjected to live cell metabolic assays using Agilent’s Seahorse XFe24 Analyzer. Extracellular acidification rates (ECARs) were measured following injection of glucose (G), oligomycin (O), FCCP (F), and antimycin A + rotenone (A+R). (B–D) Graphical representations of glycolysis (B), maximum glycolytic rate (C), and glycolytic reserve (D) are pictured (n = 12). (E) OCRs were measured following injection of glucose (G), oligomycin (O), FCCP (F), and antimycin A + rotenone (A+R). (F–I) Graphical representations of basal respiration (F), spare respiratory capacity (G), ATP-linked respiration (H), and maximal respiration (I) are pictured (n = 12). Paired t tests were used for comparisons. *P = 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig 5: Cell seeding density titrations for optimal results using fresh chicken peripheral blood mononuclear cells (PBMCs) (Ross 308 broiler and Bovans layer) in the Seahorse Xfe24 Analyzer (Agilent) by main effect of cell seeding density on (A) oxygen consumption rate (OCR) and (B) extracellular acidification rate (ECAR), and by the cell seeding density x line of bird interaction on (C) OCR and (D) ECAR. All data are presented as least square means (LSMeans1) (SEM). 1Bars that do not share letters indicate means that are significantly different (p = 0.05).