Figure 6

(A–F) PPARD downregulation in cancer cells inhibits angiogenesis in vivo. (A and B) Lung metastases were formed as described in Figure 1, A and B, harvested in optimum cutting temperature compound (OCT), and assessed for the microvessel density (MVD) marker CD31 by IHC staining. (A) Representative images of IHC staining of CD31. (B) Quantification of MVD. (C and D) Lung metastases were formed as described in Figure 2, A and B, harvested in OCT, and assessed for CD31 by IHC staining. (C) Representative images of IHC staining of CD31. (D) Quantification of MVD. (E and F) Lung metastases were formed as described in Figure 5, harvested in OCT, and assessed for CD31 by IHC staining. (E) Representative images of IHC staining of CD31. (F) Quantification of MVD. Green arrows indicate tumor blood vessels. Values in B, D, and F are mean ± SEM; *P < 0.01; ^#P < 0.001 compared with control-shRNA group. P values were calculated by 1-way (B and D) or 2-way ANOVA (F). (G and H) PPARD overexpression in colon cancer cells promotes angiogenesis in vitro. HCT116 cells stably transfected with PPARD vector or control vector were cultured in serum-free medium for 48 hours, and the conditioned media were collected for tubule formation assay using HUVECs. (G) Representative images of tubule formation. (H) Quantification of tubule formation. (I and J) The PPARD agonist GW0742 increases tubule formation by HUVECs. HCT116 cells were treated with the PPARD agonist GW0742 (1 μM) or the control solvent (DMSO) in serum-free medium for 72 hours, and the conditioned media were collected for the tubule formation assay. (I) Representative images of tubule formation. (J) Quantification of tubule formation. The values in H and J are mean ± SEM. *P < 0.01; **P < 0.001 (unpaired t test). All scale bars: 100 μm.