Extended Data Fig. 5: Regulatory mechanisms for the rapid shift towards pentose cycle (Related to Fig. 4).
a. Lactate labeling from 1,2-13C glucose after 30 minutes stimulation with various doses of PMA, with or without NOX inhibitor DPI (10 µM). Due to limited neutrophil yield from each blood draw, the entire span of dose-response curve was covered from two different donors (indicated by color code), with several overlapping conditions to ensure the trend and main observations are consistent across donors. Bar graphs present the mean value of technical duplicates. b. G6PD protein level in unstimulated, PMA (100 nM, 30 min)-stimulated, and PMA (100 nM) + DPI (10 μM) treated HL-60 cells. Results confirmed in two independent experiments (see source data). c. Relative NADPH producing rate measured in cell lysates of unstimulated and PMA (100 nM)- stimulated HL-60 cells, when cells were given G6P or F6P as substrate. Relative rate is normalized to the NADPH production in unstimulated cell lysate with G6P as substrate. Results have been repeated in two independent experiments. d. Relative redox ratio of NADPH/NADP+ is depleted during oxidative burst induced by various stimuli. NADPH and NADP+ were measured in human blood neutrophils stimulated with fMLP (100 nM) for 10 min, or Zymosan A (300 μg/mL), TNFα (100 ng/mL), or Pseudomonas aeruginosa (10:1 PsA:neutrophil) for 30 min. Nd. Indicates NADPH was not detectable. e. Treatment of electron accepter Phenazine Methosulfate (PMS) (10 μM) causes accumulation of PPP intermediates, increase in NADP + , and depletion of NADPH in neutrophils that do not engage in oxidative burst. These changes were seen either in unstimulated human neutrophils or PMA (100 nM)-stimulated neutrophils treated with 10 μM DPI to eliminate oxidative burst. f. PFK and aldolase enzyme activity measured in cell lysates of unstimulated- and PMA (100 nM) stimulated- HL-60 cells. Lysates were given substrates F6P and ATP, and the production rate of FBP and DHAP were measured and normalized to unstimulated condition. c-f. Bar graph present mean value of N = 2 technical replicates (indicated by dots) g. The level of TIGAR in HL-60 cells that are unstimulated, stimulated with 100 nM PMA for 30 min, or stimulated with 100 nM PMA and treated with DPI. Results have been confirmed in two independent experiments (see source data).
