Paxilline br Expression constructs br The A
2.7. Expression constructs
The A20 plasmids were purchased from Addgene (http://www. addgene.org/) and subcloned into pCMV-Myc and pCMV-Flag expres-sion vectors. A20 N-terminal DUB domain, C-terminal E3 ligase (ZnF1- 7) domain and their corresponding mutant structures as described were also subcloned into pCMV-Myc and pCMV-Flag expression vectors .
ERα was subcloned into pCMV-Flag, pCMV-Myc and pCMV-Flag-HA (FH) expression vectors.
2.8. Gene transfection and real-time PCR
Plasmids and siRNAs were transfected using Lipofectamine 3000 and RNAiMAX (Invitrogen) according to the manufacturer's protocol. siRNA sequences were listed in Supplementary Table S3. The mRNA levels were measured by real-time PCR (RT-PCR) using SYBR Premix Ex Taq (Takara, China). Primers were listed in Supplementary Table S4.
2.9. Drug intervention
The following drugs were used for cell interventions as indicated: phorbol-12-myristate-13-acetate (PMA) (Sigma Aldrich, P8139), IL4 (Sigma Aldrich, SRP3093), IL13 (Sigma Aldrich, SRP3274), re-combinant human IL1α (Novoprotein, C070), recombinant human IL6 (Novoprotein, C027), recombinant human IL10 (Novoprotein, C027), recombinant human MCP2 (Novoprotein, C063), recombinant human GM-CSF (Novoprotein, C003), recombinant human IL17A (Sigma Aldrich, SRP3080), IL17A receptor antagonist (R&D systems, MAB177), pyrrolidine dithiocarbamate (PDTC) (Sigma Aldrich, P8765), MG132 (Selleckchem Houston, USA, S2619), 17β-estrogen (Sigma Aldrich, E2758), cyclohexamide (CHX) (Enzo, ALX-380269), and vehicles in-cluded ethanol (EtOH), dimethyl sulfoxide (DMSO), and PBS. Prior to E2 intervention, Paxilline were cultured in phenol red-free medium sup-plemented with 10% charcoal-stripped FBS.
2.10. ESR1 dual-luciferase reporter assays
Cells were transfected with pGL3-ESR1 promoter, Firefly plasmid, and pGL4.74 [hRluc/TK] Renilla plasmid, which acted as the internal control. The pGL3-ESR1 promoter plasmids contain 4000 bp upstream of the transcription initiation site. The cells were lysed to measure the luciferase activity by VARIOSKAN FLASH (Thermo Scientific) using a dual luciferase reporter kit (Promega, E1910).
The supernatants of THP-1, CD163+ Mø, HEC-1A cells and CD163+ Mø and HEC-1A co-culture were collected for cytokine arrays (RayBio Human Inflammation Antibody Array G3, Guangzhou, Shanghai). Forty-two cytokines and their fluorescent values were described in detail in Supplementary Table S5.
2.12. Immunofluorescent staining
Cell immunofluorescent staining was performed as previously de-scribed . Primary antibodies against A20 (Santa Cruz Bio-technology, sc-166692) and ERα (Sigma Aldrich, SAB4500812) were incubated with cells overnight at 4 °C followed by incubation with secondary fluorescent-tagged antibody (anti-mouse, Alexa Fluor® 488 and 555, Life Technology) for 1 h. Nuclei were stained with 4′,6-dia-midino-2-phenylindole (DAPI) (Beyotime, China) for 5 min. Fluores-cence was detected using a fluorescence microscope (Nikon).
2.13. Co-immunoprecipitation (Co-IP) and western blotting (WB)
2.14. In vitro ubiquitination assays
In vitro ubiquitination assays were performed as previously de-scribed . HEK-293T cells were lysed with 0.5% NP40 buﬀer and then incubated with anti-Myc aﬃnity gel (Selleck, B23401) or anti-FLAG antibody-conjugated M2 agarose beads (Sigma) overnight at 4 °C. Ubiquitination levels of ERα were analyzed by western blotting.
2.15. Cell proliferation and apoptosis
5-Ethnyl-2‘-deoxyuridine (Edu) staining, CCK8 (DOJINDO, Japan) and cell apoptosis (DOJINDO, Japan) were performedas described [21,27,28].
All experiments were repeated at least three times in this study. SPSS 19.0 (IBM SPSS Software) was used for statistical analyses. Student's t-test, one-way or two-way ANOVA, and Spearman's correla-tion analysis were used for further analyses. P-values of 0.05 were considered statistically significant. Error bars indicate standard devia-tion (SD) in the graphs.