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

FIG 4.

FIG 4

EAF2 inhibits HIF-1α target gene expression. (A) Semiquantitative real-time PCR analysis showed that the expression of SLC2A (Glut-1) was reduced in EAF2 wild-type MEFs (EAF2+/+) compared to that in EAF2-null MEFs (EAF2−/−) under normoxic and hypoxic conditions. (B) Knockdown of EAF2 in HepG2 cells enhanced HIF-1α target gene expression. (B1) Semiquantitative real-time PCR analysis showed that the expression of SLC2A was enhanced in EAF2 knockdown HepG2 cells under normoxic and hypoxic conditions. (B2) Semiquantitative real-time PCR analysis showed that the expression of LDHA was enhanced in EAF2 knockdown HepG2 cells under hypoxic conditions. (C) Overexpression of EAF2 in RCC4 cells reduced expression of the HIF-1α target genes SLC2A (Glut-1) (C1), PDK-1 (C2), and LDHA (C3) under normoxic conditions, as determined by semiquantitative real-time PCR analysis. (D) Knockdown of EAF2 in RCC4 cells enhanced expression of The HIF-1α target genes SLC2A (Glut-1) (D1) and LDHA (D2) under normoxic conditions, as determined by semiquantitative real-time PCR analysis. (E) Overexpression of EAF2 in 786-O cells did not alter levels of the HIF-2α target genes PAI-1 (E1), CITED2 (E2), POU5F1 (C3), and CST (E4) under normoxic conditions, as determined by semiquantitative real-time PCR analysis. (F) Knockdown of EAF2 in 786-O cells mediated by EAF2 shRNA did not enhance expressions of the HIF-2α target genes PAI-1 (F1) and CITED2 (F2) under normoxic conditions, as determined by semiquantitative real-time PCR analysis. (G1) The BNIP3L protein level was decreased when EAF2 was overexpressed in RCC4 cells. (G2) Overexpression of EAF2 in 780-O cells did not affect HIF-2α downstream target SOD2 protein level; overexpression of pVHL was used as a positive control. (G3) Knockdown of EAF2 in RCC4 cells enhanced HIF-1α target LDHA protein level but did not affect HIF-2α target PAI-1 protein level. (G4) Knockdown of EAF2 in 786-O cells did not affect HIF-2α target PAI-1 and SOD2 protein levels. (G5) When HIF-1α was kept intact in RCC4 cells, overexpression of EAF2 reduced expression of HIF-1α target LDHA (left two lanes), but when HIF-1α was knocked down by HIF-1α shRNA in RCC4 cells, overexpression of EAF2 did not affect HIF-1α target LDHA protein level (right two lanes).