Extended Data Fig. 4: Obesity is associated with enhanced ROS in neutrophils.

a, Flow cytometry gating strategy for neutrophils, monocytes, and macrophages in lung. Gates were first drawn around CD45⁺ CD11b⁺ cells (not shown), followed by gating according to Ly6G and Ly6C as shown. CD45⁺ CD11b⁺ Ly6G⁺ Ly6C^lo neutrophils were used for RNA-seq, corresponding to Fig. 4a-d. b, Weights of LF (n = 3 mice) and HF (n = 4 mice) mice used for lung neutrophil RNA-sequencing. Mean ± SEM. c, Neutrophil CellROX flow cytometry plot, corresponding to Fig. 4e. d, Quantification of CellROX⁺ neutrophils (CD45⁺ CD11b⁺ Ly6G⁺ Ly6C^lo) in lungs from LF or HF B16F10 primary tumor-bearing mice. n = 8 mice per condition; mean ± SEM; two-tailed Mann-Whitney test. e, Quantification of CellROX⁺ monocytes (CD45⁺ CD11b⁺ Ly6G^- Ly6C^hi) in lungs from LF or HF Py230 primary tumor-bearing mice. LF, n = 5 mice; HF, n = 7 mice; mean ± SEM; two-tailed Mann-Whitney test. f, Quantification of CellROX⁺ macrophages (CD45⁺ CD11b⁺ Ly6G^- Ly6C-) in lungs from LF or HF Py230 primary tumor-bearing mice. LF, n = 5 mice; HF, n = 7 mice; mean ± SEM; two-tailed Mann-Whitney test. g, Quantification of CellROX⁺ monocytes (CD45⁺ CD11b⁺ Ly6G^- Ly6C^hi) in lungs from LF or HF B16F10 primary tumor-bearing mice. n = 8 mice per condition; mean ± SEM; two-tailed Mann-Whitney test. h, Quantification of CellROX⁺ macrophages (CD45⁺ CD11b⁺ Ly6G^- Ly6C^-) in lungs from LF or HF B16F10 primary tumor-bearing mice. n = 8 mice per condition; mean ± SEM; two-tailed Student’s t-test. i, Quantification of SOD activity (U/ml) using bone marrow neutrophils isolated from WT or ob/ob mice. LF, n = 3 mice; HF, n = 4 mice; mean ± SEM; two-tailed Mann-Whitney test. j, Quantification of SOD activity (U/ml) using serum isolated from WT or ob/ob mice. n = 5 mice per condition; mean ± SEM; two-tailed Student’s t-test. k, Quantification of MPO activity (mU/ml) using bone-marrow neutrophils isolated from LF or HF mice. n = 4 mice per condition; mean ± SEM; two-tailed Student’s t-test. l, Quantification of hypochlorite concentration in bone marrow neutrophils isolated from LF or HF mice harbouring E0771 mammary tumors (tumor-bearing; TB). LF, n = 6 mice; HF, n = 4 mice; mean ± SEM; two-tailed Student’s t-test.

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