CYP1B1^−/− ECs show higher oxidative stress with increased TSP2 expression. DHE staining of CYP1B1^+/+ (A), CYP1B1^−/− (B) ECs, and CYP1B1^+/+ ECs incubated with TMS (C) (×400) are shown. The quantitative assessment of the is shown in panel D. The data in each bar are the mean fluorescence intensities determined as described in “Methods,” and error bars indicate standard deviation. A significant increased fluorescent intensity was observed in CYP1B1^−/− cells (n = 20, *P < .05). CYP1B1^+/+ cells incubated with TMS showed higher fluorescence compared with untreated CYP1B1^+/+ cells, similar to CYP1B1^−/− cells. The level of TSP2 was analyzed by Western blotting of serum-free conditioned medium prepared from CYP1B1^+/+ and CYP1B1^−/− ECs as described in “Methods” (E). A blot of cell lysates prepared from these cells was probed with β-catenin to control for loading. Western blot analysis of whole cell lysates of CYP1B1^−/− ECs infected with the adenoviruses expressing empty vector or CYP1B1 is shown in F. Blots were probed with antibodies to TSP2, CYP1B1, and β-actin to control for loading. Please note expression of CYP1B1 in CYP1B1^−/− ECs is inversely correlated with expression of TSP2. Western blot analysis of whole cell lysates from CYP1B1^−/− ECs incubated with or without NAC for 2 days is shown in panel G. Blot was probed with TSP2, and β-actin was used as loading control. Western blot analysis of cell lysates prepared from CYP1B1^−/− ECs expressing TSP2-specific siRNAs (2574 [I] or 3611 [J]) or control siRNA, probed with anti-TSP2 or β-actin (loading control) is shown in panel H. The capillary morphogenesis of these cells in Matrigel are shown in panels I-K. The quantitative assessment of the data are shown in panel L. Capillary morphogenesis was significantly restored in CYP1B1^−/− cells expressing TSP2 siRNAs compared with control vector (n = 3, *P < .05). These experiments were repeated with 2 different preparations of ECs with similar results.