Inhibition of Tumor Angiogenesis by Phloroglucinol Figure 7. Phloroglucinol decreased the number of CD31(+) vessels a in matrigel plugs assay. (A) Representative photomicrographs of CD31-stained matrigel sections obtained from mice treated with the vehicle. After 13 days, the mice were sacrificed, and the matrigel plugs were removed. The histological sections were fixed with 4% paraformaldehyde and embedded in paraffin. Infiltrating endothelial cells in plugs stained during an immunohistochemistry assay of anti-CD31 antibody. Bar: 500 mm. (B) Quantitative assessment of CD31-positive capillaries (*P,0.05, **P,0.01). doi:10.1371/journal.pone.0033618.g007 was endothelial basal medium (EBM)-2 (Lonza, Walkersville, MD) supplemented with 5% fetal bovine serum (FBS), human basic fibroblast growth factor (bFGF), human vascular endothelial growth factor (hVEGF), human insulin-like growth factor-1 (hIGF-1), human epidermal growth factor (hEGF), ascorbic acid, and GA-1000 (complete EGM-2 medium). After 4 days, non- adherent cells were removed, and the tightly attached fraction of cells were re-plated and cultured for another 3 days. Cultivation was continued by refreshing the EGM-2 medium until spindle- shaped colonies were formed after 14–21 days. Medium was changed daily for 7 days and then every other day until the first passage. Endothelial characteristics of the attached spindle-shaped EPCs were examined. Immunohistochemistry revealed that our late EPCs (ex vivo cultured outgrowth ECs) express several endothelial lineage markers using various antibodies against CD31, VEGF receptor-2 (KDR/Flk-1), von Willebrand factor (vWF), eNOS, p-eNOS and p-Akt. . Lewis lung carcinoma (LLC) cells were obtained from College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University. The LLC cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, WelGENE) supplemented with 10% FBS and 1% penicillin/streptomycin (Gibco, Eggenstein, Germany) [40]. PLoS ONE | www.plosone.org 9 April 2012 | Volume 7 | Issue 4 | e33618