HK Inhibits Angiogenesis/Peritoneal Dissemination Figure 13. A proposed mechanism for honokiol up-regulation of endoplasmic reticulum stress, which reduced angiogenesis, growth, and peritoneal dissemination in gastric cancer cells through the calpain II/SHP-1/STAT-3 pathway. Honokiol induced the SHP- 1 activity and resultant cell STAT-3 dephosphorylation through endoplasmic reticulum stress activation and calpain-II regulation. doi:10.1371/journal.pone.0043711.g013 amount of hemoglobin assayed in parallel. The images were photographed with a Nikon digital camera, and the number of newly formed micro-vessels was counted. Ex Vivo Aortic Ring Assay Aortas were isolated from 6-week-old Sprague-Dawley rats. Plates (48-well) were coated with 120 mL of matrigel. Aortas isolated from mice were cleaned of periadventitial fat and connective tissues, and cut into ,1-mm-long to 1.5-mm-long rings. After being rinsed five times with endothelial cell–based medium, the aortas were placed on the matrigel-covered wells and covered with another 100 mL of matrigel. Honokiol or vehicle was added to the wells in a final volume of 250 mL of medium. Cultures were incubated and the media were replaced every second day over the course of the 8–10 days of experiments. Visual counts of micro-vessel outgrowths from replicate explant cultures (n$6) were done under bright-field microscopy following an established protocol The assay was scored from 0 (least positive) to 5 (most positive) in a double-blinded manner. Each data point was assayed in sextuplets. Experiments were performed at least twice, and micro-vessel counts in treated and control cultures were analyzed. Endothelial Cell Tube Formation Matrigel was thawed at 4uC overnight, and each well of prechilled 24-well plates was coated with 100 mL of matrigel and incubated at 37uC for 45 min. HUVECs (46104 cells) were added to 1 mL of ECGM with various concentrations of honokiol. After 12 h of incubation at 37uC, 5% CO2, endothelial cell tube formation was assessed with a Nikon inverted microscope with attached digital camera. Tubular structures were quantified by manual counting of low power fields (506), and the inhibition percentage was expressed using untreated wells as 100%. PLOS ONE | www.plosone.org 16 August 2012 | Volume 7 | Issue 8 | e43711