(A) Total transcripts of sorted BM, BM stimulated with Yoda1 for 2 hours (BM+Yoda1), lung, and PB neutrophils are presented in a PCA plot. n = 3. (B) BM-derived neutrophils were stimulated with Yoda1 for 2 hours. Relative mRNA expression of the indicated genes in neutrophils was determined by qPCR. n = 4. (C) BM-derived neutrophils were stimulated with Yoda1 for the indicated durations. Relative mRNA expression of the indicated genes in neutrophils was determined. n = 3–6. (D) Immunoblot analysis of phosphorylated ERK (pERK), pNF-κB, total ERK, and total NF-κB in BM-derived neutrophils treated with Yoda1 for indicated durations. Blots are representative of 3 independent experiments. (E) BM-derived neutrophils were pretreated with either trametinib (ERK inhibitor) or BAY 11-7082 (NF-κB inhibitor) for 30 minutes before stimulation with Yoda1 for 2 hours. Relative mRNA expression of the indicated genes in neutrophils was determined. n = 5. (F) BM-derived neutrophils from WT and Piezo1-cKO mice were treated with Yoda1 for 2 hours. Relative mRNA expression of the indicated genes in neutrophils was determined n = 4. (G) Schematic of experimental workflow. MACS, magnetic-activated cell sorting. (H) BM-derived neutrophils from the indicated mice were i.v. injected into CD45.1 recipient mice. Two hours later, the transferred neutrophils were sorted from the indicated tissue and subjected to qPCR analysis. Relative mRNA expression of the indicated genes in neutrophils was determined. n = 4. (I) Schematic of experimental workflow. stim-media, stimulated media.(J) BM-derived neutrophils were pulse treated as indicated in I. Relative mRNA expression of the indicated genes in neutrophils was determined. n = 6. Data in B, C, E, F, H, and J indicate the mean ± SEM. Statistical significance was determined by 1-way ANOVA with Tukey’s multiple-comparison test (C and E), multiple Student’s t test (F), and unpaired, 2-tailed Student’s t test (B, H, and J).