CXCL12 modulates the transcription factor RUNX3. (A) mRNA extracted from PBMos stimulated with CXCL12 (100 ng/mL) or unstimulated (control) was analyzed by microarrays. CXCL12-induced regulation of the expression of the RUNX family proteins and TGF-β receptor was compared. The ratios of the log2 intensity data of CXCL12-treated cells versus Control for TGF-β/RUNX selected genes are represented. Mean ± SD; n = 3. (B) PBMos were incubated for 6 hours in the presence (+) or absence ⁽⁻⁾ of CXCL12 (100 ng/mL), or the CXCR4 or CXCR7 antagonists AMD3100 (25 μg/mL) and CCX733 (10nM), respectively. RNA was extracted from cells and subjected to reverse transcriptase reaction and qPCR for RUNX3 and normalized to GAPDH. Fold regulation relative to control untreated cells is shown. Mean ± SD; n = 4; *P < .05. (C) Runx3 expression was analyzed by flow cytometry on PBMos (punctuated line), DCs (gray line), or macrophages (black line), by stimulation for 5 days with GM-CSF plus IL-4 or M-CSF, respectively. Control Isotype was also shown (shaded profile). A representative experiment of 3 is shown. (D) PBMos were differentiated to DCs or macrophages as described in panel C in the absence or presence of CXCL12 (100 ng/mL), during the differentiation process. RUNX3 levels were analyzed by flow cytometry. Fold regulation relative to control untreated ⁽⁻⁾ cells are shown. Mean ± SD; n = 3; *P < .05. (E) PBMos were cultured in the absence or in the presence of CXCL12 (100 ng/mL) during 5 days. Percentages of RUNX3 positive cells were analyzed by flow cytometry at the indicated times. Fold regulation relative to control untreated cells are shown. Mean ± SD; n = 9; **P < .01. (F) PBMos induced with M-CSF during 2 days were untreated ⁽⁻⁾ or treated with CXCL12 (100 ng/mL) as indicated. Western blot of total lysates was performed. Results were quantified by densitometry, normalized for differences in loading, and expressed as fold increase compared with unstimulated controls. A representative experiment out of 3 is shown. (G) PBMos were pretreated (+) with AMD3100 (25 μg/mL) or CCX733 (10nM) or untreated ⁽⁻⁾ as control. Subsequently, cells were stimulated with CXCL12 (100 ng/mL) as indicated. Western blot of total lysates of 48 hours cultured cells was performed. Results were quantified by densitometry, normalized for differences in loading, and expressed as fold increase compared with unstimulated controls. A representative experiment of 3 is shown.