Figure 3

SMYD3 binds to and stabilizes HIF1?, leading to an increase of nuclear HIF1? and enhancement of HIF1?-mediated target genes expression.A?C, qPCR analysis of GLUT1 (A), PGK1 (B), and VEGF (C) mRNA in HEK293T cells cotransfected with Myc-HIF1? or Myc empty vector (control) together with pCMV-SMYD3 or pCMV empty vector (EV) (control) for 24 h. Data show mean ± SD; Student?s two-tailed t test. ?p < 0.05, ??p < 0.01, ???p < 0.001. Data from three independent experiments. D, co-immunoprecipitation of HA-SMYD3 with Myc-HIF1?. HEK293T cells were co-transfected with indicated plasmids for 24 h. Anti-HA antibody-conjugated agarose beads were used for immunoprecipitation, and the interaction was detected by immunoblotting with the indicated antibodies. E, endogenous interaction between Smyd3 and Hif1?. Smyd3-deficient or wildtype MEF cells (Smyd3^?/? or Smyd3^+/+) under hypoxia for 4 h and anti-HIF1? antibody was used for immunoprecipitation. F, immunoblotting of exogenous Myc-HIF1? expression in H1299 cells transfected with an increasing amount of HA-SMYD3 expression plasmid (HA empty vector [-] was used as a control). G, immunoblotting of endogenous HIF1? expression in Smyd3-deficient or wildtype MEF cells (Smyd3^?/? or Smyd3^+/+) under normoxia (21% O₂) or hypoxia (1% O₂) for 4 h. The relative intensities of Hif1? were determined by normalizing the intensities of Hif1? to the intensities of Gapdh. Data show mean ± SD; Student?s two-tailed t test. ?p < 0.05. Data from three independent experiments. H, immunoblotting of endogenous Hif1? expression in Smyd3-deficient MEF cells reconstituted with or without wildtype Smyd3 by lentivirus under normoxia (21% O₂) or hypoxia (1% O₂) for 4 h. The relative intensities of Hif1? were determined by normalizing the intensities of Hif1? to the intensities of Gapdh. Data show mean ± SD; Student?s two-tailed t test. ??p < 0.01. Data from three independent experiments. I, Smyd3-deficient or wildtype MEF cells (Smyd3^?/? or Smyd3^+/+) were cultured under hypoxia for 4 h. Western blot analysis was used to detect Smyd3 and Hif1? in cytosol and nuclear fractions. J, confocal microscopy image of endogenous Hif1? in Smyd3-deficient or wildtype MEF cells (Smyd3^?/? or Smyd3^+/+) under hypoxia for 4 h. Scale bar = 50 ?m. MEF, mouse embryonic fibroblast; qPCR, quantitative RT?PCR; HIF, hypoxia-inducible factor.