FGF-2 down-regulates CXCL12 mRNA levels via mir-31 up-regulation. (A) Representative cultured-cell expression of Hoechst, Nestin, and internal CXCL12 in cultures of total BM cells from C57BL/6 Nestin-GFP mice treated with PBS or FGF-2 for 48 hours (100×). Scale bar indicates 20 μm. (B) Membrane-associated functional CXCL12 expression on BM Nestin⁺/CD45⁻ MSCs as determined by flow cytometry. (C) Representative flow cytometric analysis of membrane-associated functional CXCL12 levels on BM Nestin⁺/CD45⁻ MSCs. (D) mir-23a, mir-31, and mir-137 levels as determined by qRT-PCR. C57BL/6 mice were treated with either PBS or FGF-2 (n > 7). (E) mir-31 levels in total BM cells as determined by qRT-PCR. Stromal MS-5 cells were transfected with mock transfection, mir-31 mimic, nontargeting control (NTC), mir-31 inhibitor (mir-31i), or the inhibitor nontargeting control (iNTC). In some cases, cultures were treated with FGF-2. (F) CXCL12 mRNA levels as determined by qRT-PCR. HEK293T cells were cotransfected with constructs carrying either luciferase reporter gene with CXCL12 exon 3 plus the CXCL12 3′-untranslated region (UTR) in its 3′-UTR combined with nontargeting control expressing vector or the luciferase reporter gene with CXCL12 exon 3 plus the CXCL12 3′-UTR in its 3′-UTR combined with mir-31–expressing vector or the luciferase reporter gene with muted (mir-31–binding site) CXCL12 exon 3 plus the CXCL12 3′-UTR in its 3′-UTR combined with mir-31–expressing vector. (G) Measured luciferase activity is represented as the fold change relative to the control. (H) Representative tissue expression of Hoechst, Nestin, and miRNA detection probes for mir-31 scramble control (Hi), U6 control (Hii), and (Hiii-iv) mir31 in C57BL/6 Nestin-GFP mice BM (100×). Scale bar indicates 20 μm. *P < .05; **P < .01. Data shown are means ± SEM.