Figure 5. Endothelial cell (EC) cellular function was impacted by ADAR1 deficiency.

(A) ECs were isolated from the lungs of 1- to 2-wk-old inducible ADAR1 KO (i-KO) mice and cultured in vitro for cellular function assays. Tamoxifen (TM) was added to culture medium for 48 h to induce ADAR1 gene deletion before assays were performed, as shown in panel (A). (B) ADAR1 gene deletion in the tested ECs was confirmed by PCR analysis before EC function was assessed. Efficient ADAR1 gene deletion in TM-induced i-KO ECs was shown by the ratios of the deleted and floxed gene PCR products. (C) Cell growth rate was monitored by counting the cell numbers at 24-, 48-, and 72-h time points after replating the same number of the TM-treated and non-treated ECs. TM induction dramatically reduced i-KO EC numbers compared with the controls. P < 0.05, n = 9. (D, E) Proliferating ECs were monitored by Ki-67 antibody staining. The number of positively stained TM-treated i-KO cells was significantly reduced compare with the control cells. P < 0.05, n = 9. (F, G) Cell migration rate was assessed by measuring the areas covered by cells that migrated, crossing the edges from both sides (between the lines). The cell migration areas were dramatically smaller in TM-treated i-KO ECs than the controls. P < 0.05, n = 3 (control) and 9 (i-KO). (H, I) Tube formation capacity was tested on Matrigel. Far fewer tube structures developed in TM-treated i-KO ECs, and the total tube length was significantly less than the controls. P < 0.05, n = 3 (control) and 4–6 (i-KO).