Fig. 2.
Fig. 2. SDF-1 in contrast to TPO activates calcium flux, chemotaxis, trans-Matrigel migration, and MMP-9 production in normal human αIIbβ3+ cells. / (A) Calcium flux studies of Fura-2–loaded normal human αIIbβ3+ cells. TPO (100 ng/mL) or SDF-1 (500 ng/mL) was added, and subsequently calcium flux was evaluated by a spectrophotofluorimeter. Data presented are from a representative experiment repeated 3 times and yielding similar results. (B) Normal human αIIbβ3+ cells show chemotaxis to SDF-1 but not to TPO. The experiment was repeated 3 times in duplicate. The bottom chambers contained media alone (−) or 5 to 100 ng/mL TPO or 300 ng/mL SDF-1. (C) Inhibition of SDF-1–dependent chemotaxis after preincubation of megakaryoblasts with increasing concentration of LY294002 (10 μmol/L and 30 μmol/L). The experiment was repeated 3 times in duplicate. (D) Trans-Matrigel migration of megakaryoblasts toward SDF-1 and TPO gradients and the effect of MMP inhibitors. The bottom chambers contained media alone (control, c) or 100 ng/mL each of SDF-1 or TPO. The assay was carried out twice using 4 to 5 chambers. Inhibition of trans-Matrigel migration toward SDF-1 was carried out by preincubating the megakaryoblasts for 2 hours in the absence or presence of 10 μg/mL rhTIMP-1 or rhTIMP-2. Percentages of cells that migrated after 3 hours of incubation at 37°C are shown as mean ± 1 SD. (E) MMP expression in megakaryoblasts in the presence of SDF-1 and TPO. Media conditioned by megakaryoblasts after incubation for 3 hours in the absence (control) or presence of 100 ng/mL SDF-1 or TPO were analyzed by zymography. Media conditioned by KG-1 cells were used as the standard to show the positions of the MMP-9 and MMP-2 activities in the gel. The experiment was repeated 3 times. (F) Inhibition of trans-Matrigel migration to SDF-1 after preincubation of megakaryoblasts with increasing concentration of LY294002 (10 μmol/L and 30 μmol/L). The experiment was repeated twice, using 6 chambers each. (G) MMP expression in megakaryoblasts stimulated with SDF-1 without (lane 1) or with (lane 2) LY294002. The experiment was repeated 2 times.

SDF-1 in contrast to TPO activates calcium flux, chemotaxis, trans-Matrigel migration, and MMP-9 production in normal human αIIbβ3+ cells.

(A) Calcium flux studies of Fura-2–loaded normal human αIIbβ3+ cells. TPO (100 ng/mL) or SDF-1 (500 ng/mL) was added, and subsequently calcium flux was evaluated by a spectrophotofluorimeter. Data presented are from a representative experiment repeated 3 times and yielding similar results. (B) Normal human αIIbβ3+ cells show chemotaxis to SDF-1 but not to TPO. The experiment was repeated 3 times in duplicate. The bottom chambers contained media alone (−) or 5 to 100 ng/mL TPO or 300 ng/mL SDF-1. (C) Inhibition of SDF-1–dependent chemotaxis after preincubation of megakaryoblasts with increasing concentration of LY294002 (10 μmol/L and 30 μmol/L). The experiment was repeated 3 times in duplicate. (D) Trans-Matrigel migration of megakaryoblasts toward SDF-1 and TPO gradients and the effect of MMP inhibitors. The bottom chambers contained media alone (control, c) or 100 ng/mL each of SDF-1 or TPO. The assay was carried out twice using 4 to 5 chambers. Inhibition of trans-Matrigel migration toward SDF-1 was carried out by preincubating the megakaryoblasts for 2 hours in the absence or presence of 10 μg/mL rhTIMP-1 or rhTIMP-2. Percentages of cells that migrated after 3 hours of incubation at 37°C are shown as mean ± 1 SD. (E) MMP expression in megakaryoblasts in the presence of SDF-1 and TPO. Media conditioned by megakaryoblasts after incubation for 3 hours in the absence (control) or presence of 100 ng/mL SDF-1 or TPO were analyzed by zymography. Media conditioned by KG-1 cells were used as the standard to show the positions of the MMP-9 and MMP-2 activities in the gel. The experiment was repeated 3 times. (F) Inhibition of trans-Matrigel migration to SDF-1 after preincubation of megakaryoblasts with increasing concentration of LY294002 (10 μmol/L and 30 μmol/L). The experiment was repeated twice, using 6 chambers each. (G) MMP expression in megakaryoblasts stimulated with SDF-1 without (lane 1) or with (lane 2) LY294002. The experiment was repeated 2 times.

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