Fig. 5

Hypoxia increases Slug SUMOylation. a IHC analysis of xenograft tumor section. Adjacent serial sections were stained for Slug and Ubc9 combined with HIF1α. The signal was captured by a fluorescent microscope (original magnification, × 200). b Hypoxia increased the SUMOylation of Slug. Cells were exposed to normoxia or hypoxia (1% O₂) for 4 h before harvesting. Whole cell lysates were analyzed by immunoblotting with the indicated antibodies. c The ubiquitination level of Slug under hypoxic conditions. HEK293T cells were transfected with 3xFlag-tagged Slug. After 36 h of transfection, the cells were treated with MG132 under normoxic or hypoxic conditions. Immunoprecipitation was performed using an anti-Flag antibody prior to immunoblotting with the indicated antibodies. The accumulation of CD71 was used to confirm that cells were exposed to hypoxia. d Hypoxia promoted the transcriptional repression activity of Slug. After transient transfection with the indicated gene constructs and a reporter vector driven by the E-cadherin promoter, the cells were incubated for 36 h. Before harvesting, the cells were exposed to normoxia or hypoxia and then subjected to luciferase assays. The relative luciferase activity levels were normalized to the levels of pGL4.74-TK. The data were reported as mean values ± SEM, and P values were calculated via Student’s t-test. The asterisk represents a P = 0.0006. e Slug SUMOylation did not occur via HIF1α protein activity. HEK293T cells were transfected with the indicated plasmid and then exposed to normoxia or hypoxia, followed by cell harvesting. The results of immunoblotting with the indicated antibodies are shown. The asterisk and arrowhead indicate Slug modified and not modified with SUMO-1, respectively. f A decrease in the Slug–SENP1/2 interaction in response to hypoxia. HEK293T cells were transiently transfected with the indicated plasmids and then exposed to hypoxia. Immunoprecipitation was performed using an anti-Flag antibody prior to immunoblotting with the indicated antibodies