Inhibition of Tumor Angiogenesis by Phloroglucinol subsequent gate was used to select the total CD45NEG cell population. Corresponding flow cytometric analysis was used to detect CD34POS cells in the gated CD45 NEG cell population. The EPC population was represented as CD45NEG/CD34POS cells. (C) Statistical difference between phloroglucinol after oral administration of both phloroglucinol and vehicle in LLC-tumor bearing mice. Bar graph represents marked differences in EPC frequency at day 5 after daily injection of phloroglucinol or vehicle (*P,0.05). doi:10.1371/journal.pone.0033618.g005 Reagents Phloroglucinol was purchased from Sigma-Aldrich (P1178). A solution of phloroglucinol was dissolved in dimethyl sulfoxide (DMSO, Amresco). The chemical structure of phloroglucinol is shown in Figure 1A. Cell culture of circulating EPCs and LLC cells EPCs were cultured according to a previously described technique [39]. The Ethics Review Board of the Hospital of the Pusan National University of YangSan, Korea, approved the protocols, and the experimental study was conducted in accordance with the Declaration of Helsinki. In brief, mononu- clear cells (MNCs) were isolated from human umbilical cord blood (HUCB). HUCB samples (approximately 60 ml each) were collected from fresh placentas with attached umbilical cords, using density gradient centrifugation with Ficol separating solution (Amersham). Freshly isolated MNCs were cultivated in 100 mm dishes coated with 1% gelatin (Sigma). The cultivation medium Figure 6. Phloroglucinol attenuated VEGF-dependent angiogenesis a in matrigel plugs assay. (A) Experimental protocols used in the VEGF-dependent matrigel plug assay. (B) Representative matrigel plug of each group at 13 days after injection. C57BL/6 mice (n = 5 per group) were subcutaneously injected with growth factor-reduce matrigel alone (none) or a combination of VEGF (300 ng/ml) and phloroglucinol (0.94 mg/kg and 9.4 mg/kg). VEGF loaded plugs from mice exhibited red color indicating abundant red blood cells. doi:10.1371/journal.pone.0033618.g006 PLoS ONE | www.plosone.org 8 April 2012 | Volume 7 | Issue 4 | e33618