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Volume 22, Issue 12, Pages 1608-1621 (December 2015)
Reciprocal Regulation of ERα and ERβ Stability and Activity by Diptoindonesin G Zibo Zhao, Lu Wang, Taryn James, Youngeun Jung, Ikyon Kim, Renxiang Tan, F. Michael Hoffmann, Wei Xu Chemistry & Biology Volume 22, Issue 12, Pages (December 2015) DOI: /j.chembiol Copyright © 2015 Elsevier Ltd Terms and Conditions
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Chemistry & Biology 2015 22, 1608-1621DOI: (10. 1016/j. chembiol. 2015
Copyright © 2015 Elsevier Ltd Terms and Conditions
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Figure 1 Dip G Increases ERβ Protein Level while Decreasing ERα Protein Level (A and B) Hs578T-ERαLuc- (A) and Hs578T-ERβLuc-inducible cells (B) were treated with 50 ng/ml doxycycline (Dox) for 24 hr. Cells were pretreated with DMSO or 10 μM Dip G for 2 hr. Then 20 μg/ml CHX was added at time point 0 and cells were harvested as indicated. Western blotting analysis showed ERα or ERβ expression using anti-Flag antibody. β-Actin was used as loading control. Quantification of the band intensity was performed using ImageJ software. (C) MDA-MB-468-ERβ-inducible cells (Shanle et al., 2013) were treated with dox for 24 hr followed by the treatment with E2 or Dip G. (D) MCF7 cells were retro-virally infected to express Flag-ERβ followed by 24 hr of DMSO or Dip G treatment. (E) 293T cells were transfected with 3xFLAG-ERα or 3xFLAG-ERβ plasmids followed by 24 hr of DMSO or Dip G treatment. (F and G) MCF7 (F) or T47D (G) cells were treated with 0, 1, or 10 μM Dip. (G) Western blotting showed ERα or ERβ expression using anti-Flag antibody or anti-ERα antibody. β-Actin or Hsp90 was used as loading control. (H) MCF7 cells were treated with DMSO or 10 μM Dip G for 5 days. Western blotting showed ERβ protein levels using anti-14C8 and H150 antibody. Hsp90 was used as loading control. (I) Total RNA from Hs578T-ERβLuc cells was collected for qRT-PCR to examine ESR2 gene expression levels. Error bars represent ±SD values. (J) 293T cells were transfected with VT, Flag-ERβ1, or Flag-ERβcx for 24 hr, followed by another 24 hr of Dip G treatment. Western blotting showed Flag-ERβ1 and Flag-ERβcx protein levels with anti-Flag antibody. β-Actin was used as loading control. See also Figure S1. Chemistry & Biology , DOI: ( /j.chembiol ) Copyright © 2015 Elsevier Ltd Terms and Conditions
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Figure 2 Dip G Does Not Affect Global Protein Degradation by Proteasome Hs578T-ERαLuc or Hs578T-ERβLuc cells were treated with Dox to induce ER expression. (A) Both ERα and ERβ were degraded in response to E2 stimulation. (B) Dip G blocked the degradation of ERβ induced by E2 but not ERα. (C) MG132 stabilized both ERα and ERβ. (D) Cells were treated with 0–10 μM Dip G or MG132 for 24 hr. Dip G did not cause global protein ubiquitination compared with MG132. (E) Cells were treated with Dip G or MG132 (10 μM) and harvested at the indicated time points. (F) 293T cells were treated with MG132 or Dip G. (G) Resveratrol did not increase ERβ protein levels. Western blotting analysis was performed using anti-Flag, anti-Ub, anti-cyclin D1 with β-actin, or Hsp90 as loading controls. See also Figure S2. Chemistry & Biology , DOI: ( /j.chembiol ) Copyright © 2015 Elsevier Ltd Terms and Conditions
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Figure 3 CHIP E3 Ligase Is Involved in Dip G-Regulated ERα and ERβ Protein Level Change (A) Schematic diagram showed the region of ERβ that recruited CHIP E3 ligase (Tateishi et al., 2006). (B) Flag-ERβWT or Flag-ERβNΔ37 was expressed in 293T cells. Cells were pre-treated with Dip G for 2 hr and E2 was then added for another 4 hr. Quantification of the band intensity was performed with ImageJ band scan. (C and D) 293T cells were transfected with VT, Flag-ERα or Flag-ERβ, HA-CHIP, and HA-PES1. Then, cells were treated with 10 μM Dip G for 24 hr (C) or 1 μM MG132 for 4 hr (D). Whole cell lysates were immunoprecipitated with M2 beads and western blotting was performed with anti-HA antibody to examine PES1 and CHIP co-precipitated with Flag-ERα or Flag-ERβ. (E) Vector control or Flag-CHIP, and Flag-ERβ were overexpressed in 293T cells for 24 hr. 10 μM Dip G was added to the medium for another 24 hr. The protein levels of Flag-ERβ, Flag-CHIP, and endogenous CHIP were examined by western blotting with Hsp90 as the loading control. (F) MCF7-shCtrl and MCF7-shCHIP-H6 stable cells were treated with vehicle or 10 μM Dip G for 24 hr. The protein levels of Flag-ERβ and endogenous CHIP were examined by western blotting with β-actin as the loading control. (G and H) Endogenous CHIP E3 ligase was knocked down by shCHIP-H6 in (G) Hs578T-ERα- or (H) Hs578T-ERβ-inducible cells. CHX was added at t = 0. Protein levels of ER were examined using anti-Flag antibody. Knockdown efficiency of CHIP was confirmed by anti-CHIP antibody. See also Figures S3, S4, and S5. Chemistry & Biology , DOI: ( /j.chembiol ) Copyright © 2015 Elsevier Ltd Terms and Conditions
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Figure 4 Mapping of ER Interacting Domains in CHIP E3 Ligase and Molecular Docking of Dip G to CHIP (A and B) ERα (A) and ERβ (B) bind to both the U-Box and TPR domain of CHIP. 293T cells were transfected with VT, WT, ΔU-Box, or ΔTPR of HA-CHIP E3 ligase with (A) Flag-ERα or (B) Flag-ERβ. Immunoprecipitation was performed using M2 beads to pull down Flag-ER. The immunoprecipitated CHIP was examined with anti-HA antibody. (C) The ligand Dip G and the receptor CHIP E3 ligase (PDB: 2C2L) (Zhang et al., 2005) were prepared with SYBYL; and blind docking was performed using PyMOL and autodock4.2. Three best-energy results of blind-docking CHIP E3 ligase in complex with Dip G were shown, including the TPR and U-box domains of CHIP. The free binding energy was calculated and is shown below each diagram. (D and E) 293T cells were transfected with ΔU-Box or ΔTPR of HA-CHIP E3 ligase with (D) Flag-ERα or (E) Flag-ERβ. Twenty-four hours after transfection, 10 μM Dip G was added for another 24 hr. Immunoprecipitation was performed using M2 beads to pull down Flag-ER. The immunoprecipitated truncated CHIP proteins were examined with anti-HA antibody. (F) Hs578T-ER-inducible cells were treated with DMSO, 10 μM Dip G, or 1 μM MG132 for 24 hr in the presence or absence of 250 nM 17-DMAG. Flag-ERα and ERβ protein levels were examined by western blotting with β-actin as the loading control. Chemistry & Biology , DOI: ( /j.chembiol ) Copyright © 2015 Elsevier Ltd Terms and Conditions
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Figure 5 The Structure-Activity Relationship Analysis of Dip G
(A) The activity of Dip G analogs was examined after 24 hr of treatment (10 μM) in Hs578T-ER-inducible cells (Dipto-Y-03 = Dip G). Quantification of band intensity was performed with ImageJ. (B) Endogenous ERα protein levels were examined after 24 hr of 10 μM Dip G analogs treatment in MCF7 cells. (C and D) Total ubiquitination, PARP, p21, and γ-H2AX were examined in Hs578T-ERα (C) or Hs578T-ERβ (D) by western blotting analysis after cells were treated with 10 μM Dip G analogs with MG132 as a positive control. See also Table S1. Chemistry & Biology , DOI: ( /j.chembiol ) Copyright © 2015 Elsevier Ltd Terms and Conditions
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Figure 6 Dip G Differentially Affects ERα- and ERβ-Mediated Gene Expression (A) MCF7 cells were cultured in stripped media for 3 days. After pre-treatment of 10 μM Dip G for 2 hr, 10 nM E2 was added for another 24 hr. Total RNA was collected for qRT-PCR to examine the relative expression of ERα target genes including IGFPB4, GREB1, PTGES, EGR3, and CCNG1. (B and C) MDA-MB-468 or MCF7 cells were infected with retroviruses expressing GFP or Flag-ERβ, and stable cells were generated with 1 week of G418 selection. (B) Vehicle control or 10 μM Dip G were added to MDA-MB-468 cells for 24 hr. Total RNA was collected for qRT-PCR to examine the relative expression of ERβ target gene changes including CDKN1A, WNT4, and PIM1. (C) MCF7 cells were pre-treated with 10 μM Dip G for 24 hr followed by 4 hr of treatment with 10 nM E2. Total RNA was collected for qRT-PCR to examine the relative expression of GREB1. The error bars represent ±SD values. The # symbol indicates statistical significance compared with “Flag-ERβ DMSO.” ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < See also Figure S4. Chemistry & Biology , DOI: ( /j.chembiol ) Copyright © 2015 Elsevier Ltd Terms and Conditions
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Figure 7 Dip G Inhibits E2-Induced Cell Proliferation while Augmenting the Growth Inhibitory Effect of ERβ (A) MCF7 cells were cultured in stripped medium for 3 days. Cells were treated with 10 μM Dip G or 100 nM ICI 182,780 in the presence or absence of 10 nM E2, and the medium was changed every 2 days. Cell proliferation was measured by MTT assay. (B) MCF7 cells were cultured in stripped medium for 3 days. Cells were treated with DMSO, 10 nM E2, 100 nM tamoxifen, 10 μM Dip G, or in combination, and the medium was changed every 2 days. Cell proliferation was measured by cell counting. (C and D) MDA-MB-468 cells (C) or Hs578T-ERβLuc cells (D) were cultured in stripped medium for 3 days. Cells were seeded in 96-well plates at 5,000 cells/well with Dox treatment. Twenty-four hours later, cells were treated with indicated concentrations of Dip G. MTT assay was performed after 5 days of treatment. (E) Hs578T-ERβLuc cells were cultured in normal medium. Cells were treated with 50 ng/ml Dox for 24 hr. Dip G (0 or 5 μM) was added and the medium was changed every 4 days. The colony formation assay was performed after 2 weeks of treatment. Experiments were performed in duplicate. The error bars represent ±SD values. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < See also Figure S5. Chemistry & Biology , DOI: ( /j.chembiol ) Copyright © 2015 Elsevier Ltd Terms and Conditions
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