Fig. 5. Loss of the C1P-cPLA₂α interaction in primary neutrophils suppresses the pentose phosphate pathway.

(A) Quantification of pentose phosphate pathway (PPP) components Sucla2, PGLS, Cope, Tmx1, and Ebp in unchallenged cPLA₂α^+/+ and cPLA₂α^KI/KI primary neutrophils from unbiased proteomics analysis. LFQ, label-free quantification. Unpaired t-test with Welch’s correction; *p<0.05, **p<0.01, n=5 samples from 5 different mice for each genotype obtained on two separate occasions. (B) In the oxidative PPP, G6PD utilizes glucose-6-phosphate to generate NADPH and 6-phosphoglucolactone, which gluconolactolase (PGLS) converts to 6-phosphogluconate. PGD utilizes 6-phosphogluconate to generate ribulose-5-phosphate and NADPH. (C) Quantification of and immunoblotting for PGD and G6PD in protein extracts from cPLA₂α^+/+ and cPLA₂α^KI/KI primary neutrophils treated with DMSO vehicle, 5-HETE, or MK886 for 2 hours. FC to WT POI = fold change relative to wild-type protein of interest. POI was further quantified relative to the loading control (HSP90). (D) Enzyme activity assays for PGD and G6PD using protein extracts from cPLA₂α^+/+ and cPLA₂α^KI/KI primary neutrophils treated with DMSO vehicle or physcion. (E) NTEM of cPLA₂α^+/+ and cPLA₂α^KI/KI primary neutrophils treated with DMSO vehicle, physcion, or a combination of physcion plus 5-HETE. All data are displayed as boxplots (C-E analyzed by Two-way ANOVA with Šídák’s multiple comparisons; *p<0.05, **p<0.01, ****p<0.0001; n=3 to 4 biological replicates/group repeated on two separate occasions for each genotype.