Extended Data Figure 10. Breast cancer cell growth, proliferation and self-renewal are not directly affected by treatment with the Alox5 inhibitor Zil.

a, b, Neutrophils were isolated from the lungs of MMTVPyMT mammary tumour-bearing mice treated daily with Zil and used to condition culture medium (LuN-Zil) (a). Enzyme-immunoassay analysis of LTB4 levels in control, LuN or LuN-Zil medium (n = 4 (control), n = 4 (LuN), n = 3 (LuN-Zil)) (b). c, d, f–i, Analysis of CD11b⁺Ly6G⁺ neutrophils in the lung by flow cytometry (c, f, h) and primary tumour burden (d, g, i) at the time of analysis of Rag1-null mice orthotopically transplanted and intravenously injected with GFP-labelled 10⁵ primary MMTV-PyMT cancer cells (n = 3 (DMSO), n = 9 (PyMT DMSO), n = 7 (PyMT Zil)) (c, d), 10⁵ mouse 4T1 cancer cells (n = 4 (DMSO), n = 5 (4T1 DMSO), n = 7 (4T1 Zil)) (f, g) or 10⁶ human MDA-MB-231 cancer cells (n = 4 (DMSO), n = 6 (MDA231 DMSO), n = 5 (MDA231 Zil)) (h, i), and treated with Zil to complement Fig. 4d–k. e, Determination of in vivo cancer cell proliferation 18 h after intravenous injection of 10⁵ GFP-labelled MMTV-PyMT cancer cells into MMTV-PyMT tumour-bearing, Zil-treated mice by 6 h BrdU pulse and flow cytometric quantification of BrdU⁺ among GFP⁺ cancer cells in the lung (n = 3 (PyMT DMSO), n = 4 (PyMT Zil). j, Quantification of mammary tumour load of control (DMSO) or Zil-treated wild-type mice 4–6 weeks after orthotopic transplantation with 10⁶ MMTV-PyMT cells onto the mammary gland. Daily Zil treatment started 1 day prior to mammary tumour engraftment (n = 11 (DMSO), n = 8 (Zil)). k, Flow cytometric quantification of BrdU incorporation after a 3 h pulse of two primary MMTV-PyMT cell preparations and one culture of the mouse 4T1 cell line treated with 1μM Zil for 24 h in adherent conditions. l, Flow cytometric quantification of frequency of CD24⁺CD90⁺ MICs in total MMTV-PyMT cells after 3-day treatment with 1μM Zil or control DMSO in adherent culture (n = 3 per group). m, Sphere formation of MMTV-PyMT cancer cells in the presence of 1μM Zil after 7 days (technical replicate n = 8 per group of biological duplicates). Statistical analysis by two-sided t-test (b–d, f–m) and one sided t-test (e). Data are represented as mean ± s.e.m. NS, not significant, *P < 0.05, ***P < 0.001.