USP33 Deubiquitinates and Stabilizes HIF-2alpha to Promote Hypoxia Response in Glioma Stem Cells (2022)
Figure 3

Figure 3. USP33 interacts with and deubiquitinates HIF2α in GSCs.

A, B Co-immunoprecipitation (Co-IP) to detect the interaction between endogenous HIF2α and USP33 in GSCs in response to low oxygen. GSCs were cultured under 20% or 5% oxygen for 24 hours before harvest for the Co-IP. Cell lysate was subjected to immunoprecipitation with anti-HIF2α (A) or anti-USP33 (B) antibodies. Co-immunoprecipitated products were immunoblotted with the indicated antibodies. An increased interaction between HIF2α and USP33 was detected in GSCs cultured under 5% oxygen relative to 20% oxygen.

C, D Co-immunoprecipitation (Co-IP) to detect the interaction between endogenous HIF2α and USP33 in GSCs in response to CoCl2 treatment. GSCs were treated with CoCl2 (300 µM) for 12 hours before harvest for the Co-IP. Cell lysate was subjected to immunoprecipitation with anti-HIF2α (C) or anti-USP33 (D) antibodies. Co-immunoprecipitated products were immunoblotted with the indicated antibodies. An increased interaction between HIF2α and USP33 was detected in GSCs treated with CoCl2 mimicking hypoxia.

E Immunoblot analysis of the poly-ubiquitination of endogenous HIF2α proteins in GSCs with or without ectopic USP33 expression. Flag-tagged USP33 was introduced into GSCs through lentiviral infection. Forty-eight hours post infection, GSCs were treated with MG132 (20μM) for 6 hours and then harvested for ubiquitination assays. Cell lysate was subjected to immunoprecipitation with anti-HIF2α antibodies followed by immunoblot with anti-Ub antibodies. Ectopic expression of USP33 resulted in a remarkable decrease of poly-ubiquitination of HIF2α in GSCs cultured under 20% oxygen.

F Immunoblot analysis of the poly-ubiquitination of endogenous HIF2α proteins in GSCs expressing shUSP33 or shNT in response to low oxygen. Twenty-four hours post lentiviral infection, GSCs were incubated in 5% oxygen for 24 hours before harvest. MG132 (20μM) was added to cell culture 6 hours before harvest for the ubiquitination assay. Cell lysate was subjected to immunoprecipitation with anti-HIF2α antibodies followed by immunoblot with anti-Ub antibodies. Disruption of USP33 elevated poly-ubiquitination of HIF2α in GSCs cultured under 5% oxygen.

G Co-immunoprecipitation to detect the interaction between HIF2α and the wild-type (WT) or the catalytically dead (CD) USP33. Ectopic HA-tagged HIF2α were expressed in 293T cells with GFP-tagged wild-type or catalytically dead USP33. Cell lysates were subjected to immunoprecipitation with anti-HA antibodies. Co-immunoprecipitated products immunoblotted with the indicated antibodies. The catalytically dead USP33 relative to the wild type USP33 showed a stronger affinity to HIF2α.

H Co-immunoprecipitation to determine the interaction between USP33 and the wild type (WT) HIF2α or the P405A-P531A (2dPA) HIF2α mutant that lacks hydroxylation. Ectopic GFP-tagged USP33 and HA-tagged WT or 2dPA HIF2α were expressed in 293T cells. Cell lysates were subjected to immunoprecipitation with anti-HA antibodies. Co-immunoprecipitated products were immunoblotted with the indicated antibodies. The 2dPA HIF2α mutant relative to the wild type HIF2α showed a weaker affinity to USP33.