Fig. 3: USP22 interacts with ZEB1, and USP22/ZEB1 is recruited to the ZEB1-binding elements of VEGFA promoter region in HCC cell lines.

A The endogenous interaction between USP22 and ZEB1 in HCCLM3 cells verified by Co-Immunoprecipitation. B Diagram of full-length (FL) and truncated mutants of USP22. C Exogenous USP22 or its truncated mutants interact with ZEB1 in HEK293 cells. D Identification of binding domains in USP22 for ZEB1 interaction. ZEB1 protein was synthesized by transcription and translation kit in vitro. Bound proteins were analyzed by western blot. GST and GST- USP22 deletion mutants were stained by Coomassie brilliant blue staining. *, position of GST and GST- USP22 deletion mutants. E The Immunofluorescence confocal experiments were used to identify the localizations of FLAG-USP22 (green) and ZEB1 (red) in HCCLM3 cells. HCCLM3 cell was transfected with FLAG-USP22 expression plasmid for 24 h before harvested. Nucleus was stained by DAPI (blue), scale bars, 15 μm. The colocation coefficient was analyzed and calculated by Image Pro Plus software. F Schematic diagram of the predicted ZEB1 binding sites on VEGFA promoter region. G USP22 were recruited to ZEB1-binding elements in VEGFA. H USP22 knockdown reduced the recruitment of ZEB1 and accumulation of levels of H2Bub on ZEB1-binding site at VEGFA-promoter I. The DNA fragments were amplified by qPCR with the primers indicated in Supplementary data (Table S3). I Effects of USP22 on the ubiquitination level of histone H2B near the five predicted ZEB1 binding sites. J Schematic diagram of PGL3-VEGFA mutant (ZEB1 binding site mutation) luciferase assay plasmid, CACCCG was replaced by AAAAAA. K USP22 failed to upregulate transcription of VEGFA without wild type ZEB1 binding site in dual luciferase assay. In histogram, the bars represent mean ± SD (n ≥ 3), P < 0.05 is considered statistically significant.