me-dependent Effects of Jasplakinolide on the Actin Distribution of Late EPCs Jasplakinolide Impaired Late EPC Adhesion by Stabilizing F-actin A recent study has reported that remodeling the actin cytoskeleton can influence the ability of cell adhesion. To test the effects of the actin rearrangement induced by jasplakinolide on late EPC adhesiveness, late EPCs were pretreated with jasplakinolide 100 nmol/l or DMSO for 1 h and then seeded on either plastic or culture surfaces coated with different ECM proteins, such as fibronectin, collagen I and laminin. After incubation for 1 h at 37uC, late EPC adhesion was observed under microscope. As shown in Fig. 5A, the stabilization of actin by jasplakinolide impaired the EPC adhesion Jasplakinolide buy 946128-88-7 Affects Late EPC Function both on plastic and on ECM proteins compared with that in DMSO-treated cells. As integrins b1 and b3 affect the EPC adhesion, we next measured the expressions of these integrins on the late EPC surface by flow cytometry. The results show that the expressions of integrins b1 and b3, and in particular that of b1 integrin, were significantly decreased by the treatment with jasplakinolide. Stabilization of Actin by Jasplakinolide in Late EPCs Inhibited Migration Cell spontaneous migration is tightly coupled to actin assembly. Therefore, we tested whether the jasplakinolide treatment affects late EPC migration. To this end, confluent cells were subjected to jasplakinolide or DMSO for 1 h and a scrape wound was generated. Images were captured at the beginning, and at subsequent regular intervals. A significantly decreased closure of the cell free area was seen as a consequence of the jasplakinolide treatment. As the repairing of the scratch depends on not only cell migration but also proliferation, we also determined the spontaneous migration of late EPCs by Boyden chamber assay. Late EPCs were cultured in the presence of jasplakinolide or DMSO for 1 h before being placed in the upper compartment of a Boyden chamber, the lower compartment of which contained the same medium as the upper chamber. It was seen that jasplakinolide impairs late EPC migration through the membrane, with 23% less cells passing through as compared with the DMSO treated cells. To further understand the underlying mechanism of the effects of jasplakinolide 
on the migration, we explored the expression of the CXCR4 and SDF-1 systems. The results show the decrease in the CXCR-4 and SDF-1 protein expressions of jasplakinolide-treated late EPCs. Late EPCs Treated with Jasplakinolide Displayed Impaired Tube Formation Ability Jasplakinolide Affects Late EPC Function defined tubule-like structures. Quantitative assessment of tube formation was conducted by measuring the length and area of closed capillary tube networks. A reduction in tube formation was observed in late EPCs cultured under jasplakinolide conditions compared with DMSO treated EPCs. Role of NO Signaling in Jasplakinolide Down-regulated EPCs It has been shown that NO is critical to the regulation of EPC function. To investigate the effects of jasplakinolide on NO production in late EPCs, cells were pretreated with jasplakinolide or DMSO, and the protein and cultured media were collected. eNOS phosphorylation and bioavailable NO was assessed by western blot and ELISA, respectively. It was observed that the eNOS phosphorylation was significantly increased in jasplakinolide-treated late EPCs as compared with that in DMSO-treated cells. This enhancement in
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