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. 2014 Mar;34(6):1085–1099. doi: 10.1128/MCB.00718-13

FIG 3.

FIG 3

EAF2 inhibits HIF-1α transactivity. (A) The promoter activities of four well-defined hypoxia-induced genes were suppressed by overexpression of EAF2 in HEK293T cells. (A1) Epo promoter activity was suppressed by overexpression of EAF2. (A2) VEGF promoter activity was suppressed by overexpression of EAF2. (A3) Under normoxic conditions (21% O2), the induction of the BNIP3 promoter reporter by cobalt chloride (100 μM) and DFX (100 μM) and HIF-1α overexpression in HEK293T cells were suppressed by overexpression of EAF2; the induction of BNIP3 promoter reporter by hypoxia (1% O2) was also suppressed by overexpression of EAF2. (A4) Under normoxic conditions (21% O2), the induction of the p2.1 reporter by cobalt chloride (100 μM) and DFX (100 μM) and HIF-1α overexpression in HEK293T cells were suppressed by overexpression of EAF2; the induction of the p2.1 reporter by hypoxia (1% O2) was also suppressed by overexpression of EAF2. (B) The HIF reporter activity was increased in EAF2 knockout (EAF2−/−) mouse embryonic fibroblasts (MEFs) compared with wild-type MEFs (EAF2+/+) under normoxic and hypoxic conditions. (B1) VEGF promoter reporter activity under normoxic and hypoxic conditions. (B2) Epo promoter reporter activity under normoxic and hypoxic conditions. (B3) p2.1 reporter activity under normoxic and hypoxic conditions. (B4) HIF reporter (HRE) activity under hypoxic conditions. WT, wild-type HRE reporter; MT, mutated HRE reporter. (C) The truncated mutant of EAF2 (EAF2 III-VI) without the ability to bind to HIF-1α did not suppress HIF-1α transactivity. (C1) Compared to overexpression of full-length EAF2, overexpression of the truncated EAF2 III-VI mutant in HEK293T cells did not suppress p2.1 promoter luciferase reporter activity under hypoxic conditions. (C2) Compared to overexpression of full-length EAF2, overexpression of EAF2 III-VI in EAF2-null MEFs (EAF2−/−) did not suppress p2.1 promoter luciferase reporter activity. (D) EAF2 has no effect on HIF-2α transactivity. (D1) Overexpression of EAF2 in HEK293T cells did not affect PAI-1 promoter luciferase reporter activity under hypoxic conditions. (D2) Overexpression of EAF2 in HEK293T cells did not affect SOD2 promoter luciferase reporter activity under hypoxic conditions. (D3) Overexpression of EAF2 in HEK293T cells did not affect HIF-2α induced PAI-1 promoter luciferase reporter activity under normoxic conditions. (D4) Overexpression of EAF2 in HEK293T cells did not affect HIF-2α-induced SOD2 promoter luciferase reporter activity under normoxic conditions. (E) The promoter activities of two HIF-1α downstream genes (p2.1 and BNIP3) were enhanced by knockdown of EAF2 in HEK293T cells, but the promoter activity of one HIF-2α downstream gene (SOD2) was not affected by knockdown of EAF2 in HEK293T cells under hypoxic conditions. (E1) p2.1 promoter activity was enhanced by EAF2 knockdown in HEK293T cells under normoxic and hypoxic conditions. (E2) BNIP3 promoter activity was enhanced by EAF2 knockdown in HEK293T cells under normoxic and hypoxic conditions. (E3) SOD2 promoter activity was not affected by EAF2 knockdown in HEK293T cells under normoxic and hypoxic conditions. pTK-Renilla was used as an internal control, and the luciferase activity was determined 18 to 24 h after transfection.