Fig. 4: USP22 maintains ZEB1 stability by triggering deubiquitination of ZEB1.

A, B USP22 deletion reduced ZEB1 protein expression while ectopic expression of USP22 upregulated that in HCC-derived cell lines in western blotting experiments. C USP22 had no obvious effect on mRNA expression of ZEB1 in qPCR experiments, the bars represent mean ± SD (n ≥ 3), P < 0.05 is considered statistically significant. D Western blotting analysis of ZEB1 expression in wild-type and USP22 knockdown Huh7 cells treated with MG132 (10 μM) for 8 h as indicated. E Depletion of USP22 decreases endogenous ZEB1 protein stability in HCCLM3 cells. Cells were treated with 100 μM cycloheximide (CHX) as indicated. F Gray values of ZEB1 were calculated and presented in a line chart with statistical analysis. G Ectopic expression of USP22 decreased the ubiquitination of ZEB1. Immunoprecipitation of ubiquitinated proteins from Huh7 cell extracts upon overexpression of USP22. Protein was harvested after MG132 (5 μM) treatment for 3 h and ubiquitinated ZEB1 species were detected by western blotting with anti-His. H Ubiquitination of ZEB1 was upregulated by USP22 deletion. HCCLM3 cells were transfected with USP22 siRNA. Cells were immunoprecipitated with ZEB1 and immunoblotted with anti-His. I The ubiquitination level of ZEB1 could not be reduced by deubiquitinase inactive mutant USP22(m). Cells were immunoprecipitated with ZEB1 and immunoblotted with anti-His. HEK293 cells were transfected with expression plasmid encoding ZEB1 and His-ubiquitin together with wild type USP22 or USP22(m). J–L Denaturing ubiquitination assays were performed to detect the effects of USP22 on ubiquitination levels and types of ZEB1. HCCLM3 cells were transfected with ZEB1 and FLAG-USP22 together with His-tagged ubiquitin (J). HEK293 cells were transfected with ZEB1 and FLAG-USP22 or USP22(m) together with HA-tagged ubiquitin mutants, including K0 (lysineless), K48-, K63-, and K27-linked ubiquitin as indicated (K, L). The cells were treated with MG132 (5 μM) before collected. The cell lysate was immunoprecipitated with anti-ZEB1 and immunoblotted with anti-His or anti-HA.