Phenotype of ACs treated with LCN2, and lineage differentiation potential of LCN2-treated ACs. Immunolabeling of MACs cultured in the presence or absence of LCN2. The ACs cultured under each of these conditions expressed vimentin⁺ (green) (A); α-smooth muscle actin (α-SMA⁺) (red) (B); CD105⁺ (red) (C); CD106 (green) (D); and CD90 (red) (E) (original magnification ×200). (F) Immunostaining of BM ACs that were preincubated with MSC culture medium alone for 10 days and then cultured with medium that promoted osteogenesis for 21 days. (G) Immunostaining of BM ACs that had been pretreated with LCN2 in MSC culture medium and then cultured under osteoblast-inducing conditions. Osteoblastic differentiation of MSCs was clearly enhanced by preincubation with LCN2 for 10 days; an anti-osteocalcin antibody was used to identify osteoblasts; cell nuclei were stained with DAPI. (H) The fluorescence intensity of osteocalcin was significantly increased in BM ACs pretreated with LCN2 (repeated-measures ANOVA F test; P = .0015; n = 6). (I) Immunostaining showed a greater number of adipocytes in BM ACs cultured in the MSC medium and then exposed to adipogenesis medium. (J) Immunostaining revealed that adipogenesis was diminished by pretreatment of BM ACs with LCN2 for 10 days and then cultured in adipogenesis medium. Anti-FABP4 (fatty acid–binding protein 4) antibody was used to identify adipocytes; cell nuclei were stained with DAPI. (K) Fluorescence intensity analysis revealed significantly decreased FABP4 protein expression in BM AC pretreated with LCN2 (repeated-measures ANOVA F test; P < .0001; n = 6).