Fig. 4

4T1 tumours elicit mitochondrial metabolism in neutrophils. a The percentage of neutrophils that are c-Kit⁺ in naive and 4T1-bearing Balb-c mouse tissues (n = 5 per group). b Representative mitochondrial stress tests (left) of splenic neutrophils from naive and 4T1-bearing mice (oligomycin (Oligo), Carbonylcyanide-4(trifluoromethoxy)phenylhydrazone (FCCP), rotenone (R) and antimycin A (AA). ATP synthase-dependent and maximal oxygen consumption rate (OCR) quantified from multiple experiments (right, n = 4 per group). c ATP synthase OCR that is dependent on fatty acid oxidation (FAO) was assessed using etomoxir. Data (n = 4 per group) from two independent experiments. d Median fluorescent intensity (MFI) of tetramethylrhodamine ethyl ester (TMRE) staining in splenic neutrophils from naive and tumour-bearing mice (n = 3). e Representative images of splenic neutrophils from tumour-bearing and naive mice stained with mitotracker CMXROS (100 μM) and Hoechst 33342 (3.25 μM), scale bar represents 20 μm. f Native immunoblots for mitochondrial complex I (CI, NDUFS1) Complex II (CII, SDHB) and Complex IV (CIV, MTCO1) of splenic neutrophils from naive and 4T1-bearing mice. g Representative OCR of splenic neutrophils from tumour-bearing or naive mice following stimulation with phorbol 12-myristate 13-acetate (PMA) (arrow) in the presence or absence of 2-deoxy glucose (2DG) (dotted line). Quantification of area under the curve (AUC) of 2DG-stimulated neutrophils relative to PMA alone from multiple experiments (naive n = 4, 4T1 n = 6). h MFI of 2′,7′-dichlorofluorescin diacetate (DCFDA) (5 μM) in splenic neutrophils from tumour-bearing or naive mice in response to PMA (20 min) following stimulation with the indicated compounds (n = 3). i, j NADPH and ATP levels in splenic neutrophils from tumour-bearing or naive mice following stimulation with PMA in the presence the indicated compounds. Data (n = 6) are from two independent experiments. Data were analysed by two-way ANOVA with Sidak’s (h–j) or Tukey’s (a–c) multiple comparisons indicated, or Student’s t-test (d, g). All error bars show mean ± SEM. Concentrations were: Rotenone (100 nM), Antimycin A (1 µM), 2-DG (100 mM), PMA (1 µg/ml), FCCP (660 nM), Oligomycin (1.26 µM), VAS-2870 (10 μM) and etomoxir (100 µM). p Values *<0.05, **<0.01, ***<0.001. All error bars display mean ± SEM