Figure 3

Figure 3

TRIM25 causes HIF-1α stabilization dependent on its enzymatic activity, resulting in enhanced hypoxia signaling.A, IB of overexpressed Myc-HIF-1α-DM (encoding the double mutant of HIF-1α [P402A/P564A]) in HEK293T cells co-transfected with Flag-empty vector or increasing amounts of Flag-TRIM25 expression plasmids. B, IB of endogenous HIF-1α in wild-type (TRIM25^+/+) and TRIM25-deficient (TRIM25^−/−) U251 cells treated with FG4592 (20 μM) for different time periods. C, qPCR analysis of PDK1, PGK1 and, VEGF mRNA in wild-type (TRIM25^+/+) and TRIM25-deficient (TRIM25^−/−) U251 cells treated with DMSO or FG4592 (20 μM) for 12 h. D, IB of endogenous HIF-1α in TRIM25^−/− U251 cells reconstituted with PHAGE-control or PHAGE-TRIM25 by lentivirus treated with DMSO or MG132 (20 μM) under hypoxia (1% O₂) for 4 h. E, IB of endogenous HIF-1α in TRIM25^−/− U251 cells reconstituted with PHAGE-control, PHAGE-TRIM25-WT or PHAGE-TRIM25-ΔRING by lentivirus under normoxia (21% O₂) or hypoxia (1% O₂) for 4 h. F, IB of endogenous HIF-1α in TRIM25^−/− U251 cells reconstituted with PHAGE-control, PHAGE-TRIM25-WT or PHAGE-TRIM25-C50/53S by lentivirus under normoxia (21% O₂) or hypoxia (1% O₂) for 4 h. G, qPCR analysis of PDK1, PGK1, VEGF, and GLUT1 mRNA in TRIM25^−/− U251 cells reconstituted with PHAGE control, PHAGE-TRIM25-WT or PHAGE-TRIM25-C50/53S by lentivirus under normoxia (21% O₂) or hypoxia (1% O₂) for 16 h. Data in (C and G) are shown as mean ± SD; statistical significance was determined by unpaired two-tailed Student’s t test; each point represents a technical replicate of a representative experiment from three independent experiments.