Figure 3. ATXN3 selectively functions as a HIF-2α deubiquitinase to promote tumoral PD-L1 transcription.

(A and B) A549 cells were cultured under normoxia and hypoxia (hyp) (1% pO₂) for 48 hours, and surface PD-L1 levels were analyzed by flow cytometry and quantification. (C) ATXN3 specifically interacts with HIF-2α. HA–HIF-2α expression plasmid was cotransfected with or without FLAG-ATXN3 into HEK293T cells. Their interactions were examined by co-IP with anti-FLAG antibodies and by Western blotting with anti-HA antibodies. (D) The interaction between ATXN3 and HIF-1α was tested in transfected HEK293T cells. (E) Endogenous interaction between ATXN3 and HIF-2α in A549 cells. (F) HA-ubiquitin, FLAG–HIF-2α, and Myc-ATXN3 plasmids were cotransfected into HEK293T cells. HIF-2α ubiquitination was determined by immunoprecipitation of HIF-2α with anti-FLAG antibodies and immunoblotting with anti-HA antibody. (G and H) HIF-2α was cotransfected with or without ATXN3 plasmids into HEK293T cells. The transfected cells were treated with CHX for different times. The protein levels of HIF-2α (top panel) and ATXN3 (middle panel) were analyzed by Western blotting. β-Actin was used as a loading control (bottom panel). (I and J) Immunoblot analysis of HIF-2α protein stability in WT and ATXN3-KO A549 cells. (K) ATXN3 enhances hypoxia-induced PD-L1 expression through protecting HIF-2α from ubiquitination-induced protein degradation. B: Ordinary 1-way ANOVA; H and J: 2-tailed unpaired t test. *P < 0.05, **P < 0.01, ***P < 0.001.