Fig 2: Relationship between NF-?B activation with S100A9-mediated cytokine secretion. Differentiated WT and S100A9-/- Hoxb8 cells were stimulated with 100 ng/mL of LPS for 2, 4, 6 and 12 h after pre-treatment for 30 min with 10 µM NF-?B inhibitor BAY 11-7082. CCL2, IL-6, CCL4 and TNF-a secretion was measured by ELISA and expressed as percentage of LPS-stimulated samples. The results were presented as mean ± SEM of five independent experiments. * = p < 0.05; ** = p < 0.01.

Fig 3: Effect of S100A9-/- on LPS-induced cytokine mRNA expression in differentiated Hoxb8 cells. Differentiated WT and S100A9-/- Hoxb8 cells were stimulated with 100 ng/mL LPS for 2, 4, 6 and, 12 h. Expression of Ccl2, Ccl3, Ccl4, Il-1a, Il-1ß, Il-6, Tnf-a and Cxcl2 mRNAs was assessed by qPCR. Data normalization was performed using three reference genes (Actß, B2m and Gusb) and expressed as fold induction compared to the non-stimulated control for both WT and S100A9-/-. Results are shown as mean ± SEM of three independent experiments. * = p < 0.05; ** = p < 0.01; **** = p < 0.0001.

Fig 4: Morphological and phenotypic changes in wild type (WT) and S100A9-/- Hoxb8 cells after differentiation. (a) Morphology of Hoxb8 cells after May–Grünwald Giemsa staining. Representative microscopic images of three independent experiments Hoxb8 before (day 0: d0) and after removal of estrogen (day 5: d5) for the induction of differentiation in the presence of 2% stem cell factor, scale bar: 25 µM. (b) Viability of Hoxb8 cells was determined by 7-AAD staining. 10,000 events were analyzed by flow cytometry and living cells were determined as percentage of 7-AAD-negatively labelled cells on the entire population of both WT and S100A9-/- Hoxb8 cells. Data are presented as mean of % ± standard error of mean (SEM) of three independent experiments. (c) The neutrophilic differentiation of ER-Hoxb8 progenitors. F4/80, Ly6C/6G, CD11b and CD71 surface expression on WT Hoxb8 cells are showed (histograms) before and after differentiation. Surface marker expression was analyzed by flow cytometry using specific fluorescently labeled antibodies: anti-mouse F4/80 (macrophage marker), anti-mouse Ly6C/6G (or Gr-1, mouse granulocyte antigen), anti-mouse CD11b (myeloid differentiation antigen) and anti-mouse CD71 (immature myeloid antigen, [24]) antibodies. Percentage of WT and S100A9-/- Hoxb8 cells expressing Ly6C/6G, CD11b and CD71 before and after differentiation. Data are expressed as mean ± SEM of three independent experiments. (d) S100A8 and S100A9 expression in Hoxb8 cells. Non-differentiated (d0) and differentiated (d5) Hoxb8 cells were lysed and RNA was extracted. S100a8 and S100a9 mRNA transcripts were quantified using qPCR. Data are expressed as mean ± SEM of three independent experiments and were normalized to three reference genes Actb, B2m and Gusb [25]. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001. On the right side, S100A8 and S100A9 proteins, in non-differentiated (d0) and differentiated (d5) Hoxb8 cells, were detected by Western Blot. ß-actin was used as a loading control. This Western Blot is representative of three independent experiments.

Fig 5: Effect of S100A9-/- on LPS-induced cytokine secretion in differentiated Hoxb8 cells. Differentiated WT or S100A9-/- cells were stimulated with 100 ng/mL of LPS for 2, 4, 6, 12 h. CCL2, CCL3, CCL4, and TNF-a secretion was measured by CBA analysis and IL-6 and CXCL2 secretion was quantified by ELISA. Results are presented as mean ± SEM of five independent experiments. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001.