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. 2011 Aug 30;286(43):37222–37236. doi: 10.1074/jbc.M111.294116

FIGURE 1.

FIGURE 1.

CXCL12 regulates chemotaxis and survival in human maDCs. A, DCs were allowed to migrate toward CXCL12 in Transwell assays as described under “Experimental Procedures.” Results shown represent the mean ± S.E. (error bars; n = 60). **, p < 0.001. B, DCs were suspended in 0.1% BSA/RPMI 1640 medium and then incubated either with or without CXCL12 for 40 h. a upper, DCs were fixed and stained with Hoechst 33342, and photographs were taken from representative samples. Arrowheads indicate apoptotic DCs. Lower, percentage of apoptotic DCs in RPMI plus CXCL12 is shown with respect to the percentage of apoptotic DCs in RPMI, which was given an arbitrary value of 100. Results represent the mean ± S.E. (n = 12). **, p < 0.0001. At least 200 DCs were analyzed in each experiment. b, DCs were analyzed for annexin V and propidium iodide staining by flow cytometry, and apoptotic DCs (annexin+/propidium iodide−) were quantified. Results shown represent the mean ± S.E. (n = 3). **, p < 0.01. C, flow plots show staining by anti-CXCR4 and anti-CXCR7 mAbs (clear gray histograms) versus isotype-matched control mAbs (dark gray histograms). CXCR7+ HeLa cells were used as positive controls (44). Results are representative of three different experiments performed. D, RNA preparations from human monocyte-derived immature DCs (imDCs) or maDCs were analyzed by RT-PCR and agarose gel electrophoresis. Two donors are shown. CXCR7 mRNA was either not detected or detected at low levels in each sample. Positive controls included CXCR7+ HeLa cells. GAPDH control reactions demonstrated that the substrate RNAs were intact. RNA processed in the absence of reverse transcriptase was used as negative control (Neg Cont). E, DCs suspended in RPMI 1640 medium untreated (−) or treated with AMD3100 (10 μm) or with CCX733 (10 μm) were allowed to migrate toward CXCL12 (100 ng/ml) or CCL21 (100 ng/ml) in chemotactic Transwell assays. Results represent the mean ± S.E. (n = 4). *, p < 0.01. ns denotes no significant differences. F, DCs suspended in RPMI untreated (−), treated with AMD3100 (10 μm) or with CCX733 (10 μm) were incubated for 40 h either in medium without chemokine (Control) or in medium plus CXCL12 (100 ng). Subsequently, the DCs were stained with Hoechst 33342. The number represents the percentage of apoptotic DCs under each condition. At least 200 DCs were analyzed in each experiment. Results represent the mean ± S.E. (n = 3). *, p < 0.01; **, p < 0.001.