Zhu et al. Journal of Experimental & Clinical Cancer Research 2010, 29:124 http://www.jeccr.com/content/29/1/124 Page 5 of 8 Figure 2 The proliferation, cell cycle, apoptosis, invasion of SKOV-3, ES-2 and HUVEC cells induced by hypoxia. The SKOV-3, ES-2 and HUVEC cells were cultured for 3 or 7 d in normoxia or hypoxia conditions before proliferation, cell cycle (S-phage), apopotosis and invasion detected by MTT, FCM (for cell cycle and apoptosis) and Transwell as shown in methods. A. The proliferation of three cells by MTT. B. The S- phase ratio in three cells by FCM. C. The apoptosis of three cells detected by FCM. D and E. The numbers of cells invasion through the membrane indicated by Transwell after incubated for 3 days (D) or 7 days (E). Data were shown in Mean ± S.D. from three separate experiments with the similar result. * and ** indicates P < 0.05 and P < 0.01 vs. Normoxia. HUVEC cells incubated under hypoxia, normoxia or hypoxia with Sirolimus were detected by TRAP-ELISA. As shown in Table 2, the activities of telomerase were positive in all the SKOV-3 endothelial-like cells, SKOV- 3 under normoxia or with Sirolimus. The activities of telomerase were negative in ES-2 endothelial-like cells and ES-2 with Sirolimus but positive in ES-2 under nor- moxia. As we expected, the activity of telomerase was negative in HUVEC cells. The different expression of HIF-1a, CyclinD1, VEGF, Flk-1, p53 and V-src mRNA in SKOV-3, ES-2 and HUVEC cells after incubation under hypoxia In order to elucidate the underlying mechanisms for the biological behaviors changes of the ELs by hypoxia, the mRNA expression of HIF-1a, CyclinD1, VEGF, Flk-1, p53 and V-src in SKOV-3, ES-2 and HUVEC cells incu- bated under hypoxia, normoxia or hypoxia with Siroli- mus were detected by Real-time PCR. The genes expression mentioned above in SKOV-3 and SKOV-3 relative cells were shown in Fig. 3A and Fig. 3B indi- cated the genes expression in ES-2 and ES-2 relative cells. As shown in Fig. 3, HIF-1a mRNA expression in both of the two tumors’ ELs was significantly higher than that in the cells under normoxia and with Sirolimus, and than that in HUVEC cells. VEGF mRNA expression in both of the two tumors’ ELs was significantly higher than that in the cells under normoxia and with Sirolimus, but was greatly lower than that in HUVEC cells. Flk-1 mRNA expression in both of the two tumors’ ELs was significantly higher than that in the cells under normoxia, but was greatly lower than that in HUVEC cells. On the other hand, Flk-1mRNA expression in ES- 2 endothelial-like cells was significantly higher than that in cells treated with Sirolimus, however, there was no difference in Flk-1 mRNA expression between SKOV-3 endothelial-like cells and SKOV-3 cells treated with Sirolimus.