E2F1 inactivation abolishes hypoxia-induced proliferation and DNA synthesis in PASMCs. Cells transfected with negative control siRNA (siNC) or E2F1 siRNA (siE2F1) were cultured under hypoxic (2% oxygen) or normoxic conditions for 24 h. (A) The mRNA level of E2F1, (B) the protein levels of E2F1, CCNE1, and β-actin, (C) the relative protein levels of E2F1 and CCNE1 were determined from the immunoblots by densitometric analysis. (D) Cell viability and (E) DNA synthesis were determined using the methods as described in materials and methods. Values are mean ± SEM (n=5). *P<0.05 compared with hypoxic cell transfected with negative siRNA.