CYP1B1^−/− EC fail to undergo capillary morphogenesis in Matrigel. CYP1B1 expression in mouse retinal ECs and C3H10T1/2 cells incubated with and without TCDD (A), and human retinal ECs and umbilical vein ECs (B), were evaluated by Western blot analysis of total cell lysates. The purified human recombinant CYP1B1 protein was used as positive control. β-catenin or β-actin was used as loading control. CYP1B1 or HE/PAS wholemount staining of retinal trypsin digests, prepared from CYP1B1^+/+ (right panels) and CYP1B1^−/− (left panels) are shown in panel C. Capillary morphogensis of CYP1B1^+/+ (D) and CYP1B1^−/− (E) retinal ECs were evaluated by plating the cells in Matrigel as described in “Methods.” After 17 hours of incubation, CYP1B1^+/+ ECs formed well-organized capillary-like networks, while CYP1B1^−/− ECs ability to organize was severely compromised (×40). A similar inhibition can be observed by incubating the CYP1B1^+/+ ECs with TMS (F), a specific inhibitor of CYP1B1 activity. Human retinal ECs also form well-organized capillary networks in Matrigel (G), which was attenuated in the presence of TMS (H). The quantitative assessments of the data are shown in panels I and J. Data in each bar are the mean number of branches per 5 high-power fields (×100; error bars indicate standard deviation). Please note that the mean number of branch points formed by CYP1B1^−/− ECs or CYP1B1^+/+ ECs incubated with TMS were significantly lower than those formed by CYP1B1^+/+ retinal ECs (n = 3, *P < .05). These experiments were repeated with 3 different preparations of ECs with similar results.