Figure 2. ATXN3 potentiates IFN-γ–induced PD-L1 expression through stabilizing IRF1 and STAT3.

(A and B) WT and ATXN3-KO cells were treated with IFN-γ (10 ng/mL) for 24 hours, and surface PD-L1 levels were analyzed. (C) ATXN3 interacts with IRF1 in transiently transfected HEK293T cells. (D) Interaction of endogenous ATXN3 and IRF1 in A549 cells. (E) HA-ubiquitin and FLAG-IRF1 expression plasmids were cotransfected with Myc-ATXN3 into HEK293T cells. IRF1 ubiquitination was determined by immunoprecipitation of IRF1 and immunoblotting with HA antibody. (F and G) FLAG-IRF1 was cotransfected with or without Myc-ATXN3 plasmids into HEK293T cells. The transfected cells were treated with cycloheximide (CHX) for different times. The protein levels of FLAG-IRF1 (top panel) and Myc-ATXN3 (middle panel) with β-actin control (bottom panel) were analyzed by Western blotting. Representative images (F) and quantification data from 3 independent experiments are shown (G). (H and I) Immunoblot analysis of IRF protein stability in WT and ATXN3-KO A549 cells as in F and G. (J) Interaction between ATXN3 and STAT3 in transfected HEK293T cells. (K) Endogenous interaction between ATXN3 and STAT3 in A549 cells. (L) The effect of ATXN3 on STAT3 ubiquitination was determined as in E. (M and N) The effects of ATXN3 on STAT3 protein stability were analyzed as in F and G. (O and P) Immunoblot analysis of STAT3 protein stability in WT and ATXN3-KO A549 cells as in H and I. (Q) The interaction between ATXN3 and STAT1 was tested in A549 cells. (R) ATXN3 enhances tumoral PD-L1 expression through protecting IRF1 and STAT3 from ubiquitination-induced protein degradation. B: Ordinary 1-way ANOVA; G, I, N, and P: 2-tailed unpaired t test; *P < 0.05, **P < 0.01, ***P < 0.001. WCL, whole-cell lysate.