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    r> and ERα through NF-κB signaling. Since these cytokines mainly upre-gulated ERα protein with little effect on ESR1 transcripts in EC cells, we speculated that A20-mediated post-translational modulation of ERα might be an important mechanism for increasing ERα abundance in the infiltrating macrophage-rich microenvironment of EC. 
    3.4. A20 increases ERα protein by preventing protein degradation
    To test our hypothesis that A20 upregulated ERα protein in EC Paclitaxel (Taxol) post-translationally, we compared ERα protein levels and ESR1 mRNA levels through plasmid transfection of A20 in EC cells. Overexpression
    Fig. 3. A20 and ERα are upregulated by CD163+ macrophages via cytokines. A. Co-culture of CD163+ macrophages and HEC-1A cells significantly upregulated the production of cytokines. CD163+ macrophages and HEC-1A were co-cultured for 24 h. Culture medium from this co-culture system as well as those culturing THP1 cells, CD163+ macrophages, and HEC-1A cells alone were separately extracted. Cytokines in the culture medium were detected by cytokine array. Red boxes indicate GM-CSF, IL1α, IL6, IL10, IL17A, MCP2, and TNFα, as marked in the key. B. Fluorescence values of seven cytokines. C. Dysregulated cytokines at a dose of 50 ng/ml upregulated A20 and ERα protein expression, especially IL1α, IL17A, and TNFα. HEC-1A cells were treated with these cytokines for 36 h and then harvested for western blotting. The fold change of ERα and A20 relative to control group was calculated from densitometry. D. IL1α, IL17A, and TNFα upregulated A20 and ERα expression through NF-κB signaling. HEC-1A cells were pretreated with NF-κB inhibitor 20 μM pyrrolidine dithiocarbamate (PDTC) for 4 h and then were treated with these cytokines for 36 h. E. IL1α, and TNFα upregulated A20 transcripts without affecting ERα transcripts, while IL17A upregulated ERα transcripts very slightly. HEC-1A cells were treated with these cytokines for 16 h and then harvested for RT-PCR. *P < 0.05, ***P < 0.001.
    of A20 upregulated ERα protein levels but not the corresponding ESR1 mRNA levels (Fig. 4A and B). Luciferase reporter assays in HEK-293T cells confirmed that there was no activating effect of A20 on the ESR1 promoter (Fig. 4C). Furthermore, silencing of A20 by A20-targeted siRNAs led to a decrease in ERα protein and CSTD mRNA (ERα target gene) without affecting ESR1 mRNA (Fig. 4D and E). These results all suggested that A20 might regulate ERα post-translationally. As A20 is an important ubiquitin-editing protein that regulates protein degradation or stability, we supposed that A20 might upregu-late ERα protein through prevention of ERα protein degradation. Overexpression of A20 prolonged the half-life of endogenous ERα protein and downregulation of A20 expression by using siRNAs towards A20 shortened the half-life of endogenous ERα protein in the presence of protein synthesis inhibitor CHX (Fig. 4F and G). Taken together, these findings suggest that A20 increased ERα protein stability by protecting ERα protein from degradation.
    We next investigated whether A20 stabilized ERα protein through protein interactions. Exogenous and endogenous co-IP experiments were performed to assess the binding of ERα and A20. Reciprocal co-IP assays confirmed that A20 interacted with ERα (Fig. 5A and B). Fur-thermore, endogenous A20 and ERα were co-localized in the nuclei of EC cells (Fig. 5C). These suggested that A20 might form a complex with ERα protein in cell nuclei to stabilize ERα protein. 
    3.6. A20 stabilizes ERα protein through deubiquitination of ERα protein
    As A20 was found to interact with ERα and stabilize its protein structure, experiments were conducted to determine which domain of A20 was involved. A20 N-terminal DUB domain, C-terminal E3 ligase ZnF1-7 domain, and their corresponding mutant structures were es-tablished (Fig. 6A). Co-IP assays showed that ERα could bind with the N-terminal and C-terminal of A20 and their mutant structures (Supplementary Fig. S4), but ectopically expressed A20 N-terminal domain stabilized ERα protein instead of A20 C-terminal domain (Fig. 6B). As the DUB activity of A20 is attributed to its N-terminal OTU domain, we deleted the OTU domain from A20 to further confirm our hypothesis. Deletion of the A20 OTU domain had no stabilization effect on co-transfected ERα protein in HEK-293T cells (Fig. 6C). Similarly, enzymatic dead mutant (C103A) A20 also had no stabilization effect on co-transfected ERα protein (Fig. 6D). HEC-1A cells transfected with different domains of A20 showed that only the A20 DUB domain sta-bilized ERα protein (Fig. 6E), which suggested that the A20 OTU do-main was required for ERα stabilization.