Supplementary Figure 2: The role of PU.1 during distinct neutrophil defense steps.

(a,b) Experimental setups of systemic Candida albicans (C. albicans) infections of PU.1^WT and PU.1^ΔNeu mice with a lethal (5x10⁵ colony forming units, cfu) (a) or sublethal (2x10⁵ cfu) (b) fungal dose to obtain survival curves (a) or perform time point analyses (b). (c,d) Fungal burden of brains from PU.1^WT (c: n = 5, d: n = 6) and PU.1^ΔNeu (c,d: n = 6) mice one (c) and three (d) days after C. albicans-infection (2x10⁵ cfu). Data are from two independent experiments. Small horizontal lines indicate the mean ( + s.e.m.) *P = 0.0173 (unpaired t-test, two-tailed). (e) Flow cytometry analysis of CD62L expression on the surface of peripheral blood neutrophils of PU.1^WT and PU.1^ΔNeu mice one (d1) and three (d3) days post C. albicans-infection (2x10⁵ cfu). (f) Frequency of CD62L^lo expressing neutrophils in the PB of unstimulated PU.1^WT (n = 3) and PU.1^∆Neu (n = 4) mice. Each symbol represents an individual mouse; small horizontal lines indicate the mean ( ± s.e.m.). The experiment was independently performed twice with similar results. (g) Mean fluorescence intensity (MFI) of CD11b on PB neutrophils in C. albicans-challenged (2x10⁵ cfu) PU.1^WT (n = 3) and PU.1^ΔNeu (n = 3) mice. Each symbol represents an individual mouse; small horizontal lines indicate the mean ( ± s.e.m.). (h) Ratio of neutrophil numbers between kidneys and the peripheral blood (number of kidney neutrophils divided by the number of blood neutrophils) in PU.1^WT and PU.1^ΔNeu mice (n = 3 each) three days post C. albicans-infection (2x10⁵ cfu). Data are the mean ± s.e.m. **P = 0.0036 (unpaired t-test, two-tailed). (i,j) Expression of NADPH oxidase subunit genes Cybb, Cyba, Ncf, Ncf2 and Ncf4 (i) and genes coding for immune response receptors important for C. albicans sensing: Clec7a, Clec4e and Clec4n (j) in flow-sorted neutrophils (Gr-1⁺) isolated from the BM of PU.1^WT (n = 2) and PU.1^ΔNeu mice (n = 3). Results were obtained from microarrays and are given in normalized intensity values. Small horizontal lines indicate the mean. All expression differences of the indicated genes were independently reproduced by qRT-PCR (data not shown).