Figure 2
Figure 2. LCN2 levels are elevated in PMF MNC-CM, and LCN2 is localized to MF marrow myeloid cells. (A) LCN2 levels in media conditioned with MF or PV MNCs (n = 10 and n = 7, respectively) and normal bone marrow MNCs (n = 10) after incubation in serum-free expansion medium containing stem cell factor, thrombopoietin, fms-like tyrosine kinase 3 ligand, and interleukin-3 for 1 day (Kruskal-Wallis nonparametric ANOVA; P < .0034 comparing normal vs MF and PV vs MF). (B) qRT-PCR results showed that LCN2 transcript levels were greater in MF T- and B-cell–depleted MNCs, as well as in MF CD34+ cells (repeated-measures ANOVA F test; P = .02 and P = .002, respectively; n = 5). (C) Immunofluorescence staining with an anti-LCN2 antibody showing LCN2 (green) presented in normal and MF MNCs; nuclei were stained with DAPI (original magnification ×200). A greater proportion of MF MNCs expressed LCN2 compared to normal MNCs. (D) Photomicrograph demonstrating LCN2 expression by cells within a bone marrow biopsy specimen from an MF patient by staining with a LCN2 monoclonal antibody (clone 5G5, dilution 1:25, Abcam). LCN2 was expressed by myeloid cells but not by erythroid precursors or megakaryocytes (original magnification ×400). Flow cytometric analysis of LCN2 receptor expression by CD34+ cells and normal BM MNCs (E) and MF MNCs (F). (G) A greater proportion of normal BM MNCs (n = 5) expressed the LCN2 receptor as compared to MF MNCs (n = 6) (Wilcoxon rank sum test; P = .008). (H) Similarly, a greater proportion of normal BM CD34+ cells (n = 5) expressed the LCN2 receptor as compared to MF CD34+ cells (n = 5) (Wilcoxon rank sum test; P = .012). (I) A greater proportion of normal BM CD34− cells (n = 5) expressed the LCN2 receptor as compared to MF CD34− cells (n = 5) (Wilcoxon rank sum test; P = .03). (J) qRT-PCR studies indicated that LCN2 receptor gene expression was similar in both MF and normal T- and B-cell–depleted MNC cells samples. (K) qRT-PCR studies indicated that that LCN2 receptor gene expression was similar in both MF and normal CD34+ cells. DAPI, 4,6 diamidino-2-phenylindole.

LCN2 levels are elevated in PMF MNC-CM, and LCN2 is localized to MF marrow myeloid cells. (A) LCN2 levels in media conditioned with MF or PV MNCs (n = 10 and n = 7, respectively) and normal bone marrow MNCs (n = 10) after incubation in serum-free expansion medium containing stem cell factor, thrombopoietin, fms-like tyrosine kinase 3 ligand, and interleukin-3 for 1 day (Kruskal-Wallis nonparametric ANOVA; P < .0034 comparing normal vs MF and PV vs MF). (B) qRT-PCR results showed that LCN2 transcript levels were greater in MF T- and B-cell–depleted MNCs, as well as in MF CD34+ cells (repeated-measures ANOVA F test; P = .02 and P = .002, respectively; n = 5). (C) Immunofluorescence staining with an anti-LCN2 antibody showing LCN2 (green) presented in normal and MF MNCs; nuclei were stained with DAPI (original magnification ×200). A greater proportion of MF MNCs expressed LCN2 compared to normal MNCs. (D) Photomicrograph demonstrating LCN2 expression by cells within a bone marrow biopsy specimen from an MF patient by staining with a LCN2 monoclonal antibody (clone 5G5, dilution 1:25, Abcam). LCN2 was expressed by myeloid cells but not by erythroid precursors or megakaryocytes (original magnification ×400). Flow cytometric analysis of LCN2 receptor expression by CD34+ cells and normal BM MNCs (E) and MF MNCs (F). (G) A greater proportion of normal BM MNCs (n = 5) expressed the LCN2 receptor as compared to MF MNCs (n = 6) (Wilcoxon rank sum test; P = .008). (H) Similarly, a greater proportion of normal BM CD34+ cells (n = 5) expressed the LCN2 receptor as compared to MF CD34+ cells (n = 5) (Wilcoxon rank sum test; P = .012). (I) A greater proportion of normal BM CD34− cells (n = 5) expressed the LCN2 receptor as compared to MF CD34− cells (n = 5) (Wilcoxon rank sum test; P = .03). (J) qRT-PCR studies indicated that LCN2 receptor gene expression was similar in both MF and normal T- and B-cell–depleted MNC cells samples. (K) qRT-PCR studies indicated that that LCN2 receptor gene expression was similar in both MF and normal CD34+ cells. DAPI, 4,6 diamidino-2-phenylindole.

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