Fig. 4. USP22 is a deubiquitinase for Atgl transcription factor FOXO1.

(A to E) Effect of EPI on FOXO1 protein expression in WT and USP22-null MDA-MB-231 cells without [(A) and (B)] or with MG132 treatment [(C) and (D)]. The mRNA levels of FOXO1 were analyzed by real-time RT-PCR (E). (F to I) Analysis of USP22, FOXO1, and ATGL expression levels by IHC in syngeneic tumor tissues as shown in Fig. 2D. Representative images (F) (scale bars, 50 μm) and quantification data [(G) to (I)] are shown. (J to M) FOXO1 interaction in HEK239T cells transiently transfected with FOXO1 and USP22 (J) or USP22 truncated mutants (K) expressing plasmids and in MDA-MB-231 cells (L) or further with EPI treatment (M). WB, Western blotting. WCL, whole cell lysate. (N to P) Analysis of FOXO1 ubiquitination in HEK293T cells coexpressed with USP22 (N) and its mutants (O) and in USP22 WT and KO MDA-MB-231 cells (P). (Q) Immunoblot analysis of FOXO1 protein degradation in HEK293T cells transfected with or without USP22 (n = 3). (R) Analysis of FOXO1 binding to Atgl promoter by ChIP in WT and USP22 KO MDA-MB-231 cells (n = 5). (S) Reconstitution of FOXO1 expression in USP22-null MDA-MB-231 cells restored ATGL expression. (T to Y) Analysis of FOXO1 expression in human breast cancer tissues [(T) and (U) with para-tumor tissues as controls or (V) and (W) in EPI^high and EPI^low groups] as well as its correlation with USP22 (X) and EPI (Y). Scale bars, 50 μm. Pearson correlation coefficient was used as a measure of association. Statistical significance was determined by one-way ANOVA test or unpaired Student’s t test. Data are expressed as means ± SD of three or more independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. ns, no statistical significance.