HIF-TWIST inhibits p21 expression via suppressing E2A protein level and activity. (A) Analysis for cell-cycle distribution in normoxic and hypoxic cells by propidium iodide (PI) staining followed by flow cytometric analysis. G₀/G₁ phase is reduced but S and G₂/M phases are increased in hypoxic cells compared with normoxic cells. (B) Western blotting for cell-cycle-related proteins. Hypoxic cells decrease in p21 protein level compared with normoxic cells. (C) Western blotting and (D) quantitative RT-PCR for E2A expression. After expansion, normoxic cells increase in E2A protein and mRNA levels compared with hypoxic cells. (E) Western blotting for HIF-1α and TWIST. Expression of HIF-1 and TWIST are decreased in normoxic cells compared with hypoxic cells. (F) Cell lysates were detected by Western blotting. Compared with control vectors, overexpression of TWIST in normoxic cells inhibits the expression of p21 and E2A, and siRNAs against TWIST in hypoxic cells induces p21 and E2A expression. The results shown here are representative of 3 independent experiments. Values are mean + SD; *P < .05 and **P < .01 indicate significant variance (independent t test) between normoxic and hypoxic groups.