Figure 5

DIF-1 inhibited angiogenesis in xenograft tumor. (A-I) DIF-1 suppressed human cervical carcinoma cell line (HeLa)-induced angiogenesis. HeLa cells mixed with liquid Matrigel in the presence or absence of 30 μM of DIF-1 were injected subcutaneously into the flanks of nude mice. The removed tumor sections were stained with hematoxylin-eosin staining (A-D) and immunofluorescence staining using PECAM-1(CD31) (E-H) (Higher magnification of the boxed areas in A, C, E and G are shown in B, D, F and H, respectively). The scale bars indicate 500 μm. (I) The expression of PECAM-1 (CD31) was analyzed with fluorescence microscopy and expressed as the mean strength of fluorescence in Matrigel-plug (mean ± SE) of three independent experiments. (J) DIF-1 suppressed PECAM-1 protein expression in the removed tumors. The samples of the removed tumor were subjected to Western blot analysis using the anti-PECAM-1 (CD31) antibody. The membrane was re-probed with the anti-GAPDH antibody. The levels of protein bands were quantified and are shown as percentage of the control level. Values are mean ± SE of three independent experiments. The asterisk indicates *P < 0.05, **P < 0.001 versus control.