Figure 4.

Loss of Cx37 does not alter the maturation of vessels in TC-1 tumors. (A,B) Left panels: immunostaining of desmin (A) and alpha-smooth muscle actin (αSMA; (B)) shows the mural cells which surrounded VeCad-positive EC in the newly formed vessels of TC-1 tumors. Right panels: The right bar graphs show that the coverage of vessels by mural cells was similar in the tumors grown in Cx37−/− and WT mice. Data are mean + SEM of 8–15 fields, from at least 5 mice per group. Bars = 100 μm. (C) Western blots confirmed that the tumors grown in Cx37−/− and WT mice featured similar levels of desmin and αSMA. Data are mean + SEM. n = 7 WT and 8 Cx37−/− mice (Student’s t-test). (D) Infusion of FITC-labeled tomato lectin (green) stained most CD31-positive vessels (red) of TC-1 tumors in both Cx37−/− and WT mice (white arrows indicate some of the few vessels that were not perfused by the lectin). Quantitative analysis showed that a similar proportion of tumor vessels was perfused in the two types of animals. Data are mean + SEM of 6–8 fields from 4 mice per group (Student’s t-test). (E) Immunostaining of pimonidazole (green) revealed hypoxic areas in tumor regions containing VeCad-positive vessels (red). Quantitative analysis revealed that the percentage of these hypoxic areas was comparable in the tumors grown in Cx37−/− and WT mice. Data are mean + SEM of 8–15 fields from 7 mice per group (Student’s t-test). Bars = 100 μm. (F) Western blots of total proteins extracted from TC-1 tumors confirmed comparable levels of pimonidazole-protein adducts in the tumors grown in Cx37−/− and WT mice. Total protein stain (MemCode) was used to control for a comparable protein loading. The specificity of the pimonidazole staining was assessed using tumor samples of WT and Cx37−/− mice which had not been previously injected with the pimonidazole HCl (HypoxyprobeTM) and revealed no adducts’ signal (data not shown). Data are mean + SEM. WT (n = 8) and Cx37−/− (n = 7) mice (Student’s t-test).