LCN2 promotes the proliferation of BM stromal cells that express the LCN2 receptor. (A) Immunofluorescence staining using an anti-LCN2 receptor antibody (SLC22A17) indicating that normal BM ACs express the LCN2 receptor (green). The nuclei were stained with DAPI. Images represent the analysis of ACs from 2 different donors. (B) Western blot analysis of 4 different samples of normal BM ACs indicated the expression of the LCN2 receptor. (C) Phase-contrast microscopy showing AC confluence that occurred after the culture of BM MNCs with 0, 10, 20, and 50 ng/mL of LCN2 (original magnification ×200). (D) The degree of AC proliferation was assessed with the PrestoBlue Cell Viability Reagent; cell numbers were determined on the basis of fluorescence intensity at 560 nm. LCN2 treatment led to the generation of greater numbers of ACs by normal marrow MNCs (repeated-measures ANOVA F test; P = .008; n = 7). (E) Immunofluorescence analysis of vimentin expression showed that AC proliferation was impaired by the addition of NAC to LCN2-containing cultures. Similar results were obtained with 3 additional BM MNC samples; NAC alone did not affect AC proliferation. (F) Analysis of fluorescence intensity of vimentin expression showed a significantly increased expression in ACs from BM MNCs cultured with LCN2 alone (repeated-measures ANOVA F test; P = .007; n = 4), which was not observed with the addition of NAC alone or NAC plus LCN2. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.