Supplementary Figure 4: Generation and validation of immortalized PU.1^∆Neu neutrophil progenitor lines.

(a) Experimental procedure of the generation of immortalized neutrophil progenitor cell lines from PU.1^WT and PU.1^ΔNeu mice. BM cells were isolated and enriched for progenitor cells by gradient centrifugation. Progenitor cells were transfected with the ER-Hoxb8 (HoxER) retrovirus and were cultured with supplementation of β-estradiol and stem cell factor (SCF). For neutrophil differentiation, progenitor cells were cultured without β-estradiol with supplementation of granulocyte colony-stimulating factor (G-CSF) for three to four days. To stimulate neutrophils, they were exposed to heat inactivated C. albicans cells for two hours. (b) PCR analysis of the PU.1 excision in progenitors (d0) and neutrophils (d4) of HoxER-PU.1^WT (WT) and HoxER-PU.1^ΔNeu (ΔNeu) lines. The experiment was independently performed more than five times with similar results. (c) qPCR analysis of Spi1 expression in progenitors (d0, n = 3 both genotypes) and neutrophils (d4, n = 4 both genotypes) of HoxER-PU.1^WT (WT) and HoxER-PU.1^ΔNeu (ΔNeu) lines. Results are shown as fold over actin transcripts (mean±s.e.m.) of three (d0) and four (d4) independent experiments. ***P = 0.0003 (unpaired t-test, two-tailed). (d) Immunoblot analysis of PU.1 expression in HoxER-PU.1^WT and HoxER-PU.1^ΔNeu progenitors (d0) and neutrophils (d4). Detection of valosin-containing protein (VCP) served as loading control. The experiment was independently performed twice with similar results. (e,f) Flow cytometry analysis of HoxER-PU.1^WT and HoxER-PU.1^ΔNeu progenitors (e) and neutrophils (f). The experiment was independently performed more than five times with similar results. (g) Morphological analysis of cytospins stained with Giemsa of HoxER-PU.1^WT and HoxER-PU.1^ΔNeu neutrophils. Scale bar resembles 10 µm. The experiment was independently performed twice with similar results. (h,i) Gene set enrichment analysis of gene signatures generated from the top 500 upregulated genes in HoxER-PU.1^WT (h; Top 500 HoxER-PU.1^WT) or HoxER-PU.1^ΔNeu (i; Top 500 HoxER-PU.1^ΔNeu) neutrophils obtained from mRNA-seq in flow-sorted (Gr-1⁺) neutrophils from the BM of PU.1^WT (n = 2) and PU.1^ΔNeu (n = 3) mice. Enrichment score calculations (for n = 500 genes each) are based on weighted Kolmogorov-Smirnov-like statistics, adjusted for multiple testing after Benjamini-Hochberg. (j) ROS production in HoxER-PU.1^WT (PU.1^WT; n = 3) and HoxER-PU.1^ΔNeu (PU.1^ΔNeu; n = 3) neutrophils stimulated with heat inactivated C. albicans cells. Data were obtained from three independent experiments. Each symbol represents and individual sample, small horizontal lines indicate the mean ( + s.e.m.). ***P = 0.0003, (unpaired t-test, two-tailed). (k) Distribution of expression of cluster genes (I-IV) visualized as the log₂ of the reads per kilobase per million (RPKM) values.