Journal: Oncology Letters

Article Title: Prostaglandin E2 triggers cytochrome P450 17α hydroxylase overexpression via signal transducer and activator of transcription 3 phosphorylation and promotes invasion in endometrial cancer

doi: 10.3892/ol.2018.9165

Figure Lengend Snippet: High expression of CYP17 promotes the secretion of androgens. (A) ELISA for estrogen, androgen and progesterone concentrations in PE cells, Ishikawa cells (control) and PGE2-stimulated Ishikawa cells. All cells were cultured for 48 h. Reverse transcription-quantitative polymerase chain reaction analysis for Ishikawa cells (B) following transfection of siCYP17 and (C) in the CYP17 OE group. (D) Western blot analysis of Ishikawa cells in control groups, siCYP17 groups and CYP17 OE groups. (E) ELISA was performed to determine the concentration of androgens in PE cells, Ishikawa cells (control), PGE2-stimulated Ishikawa cells and CYP17 OE Ishikawa cells. All cells were cultured for 48 h. *P<0.05, **P<0.01. CYP17, cytochrome P450 17α hydroxylase; PE, primary endothelial; siCYP17, CYP17 small interfering RNA; CYP17 OE, CYP17 overexpression.

Article Snippet: The ELISA kits for estrogen (cat. no. CEA461Ge, 96T), androgen (testosterone, cat. no. CEA458Ge, 96T) and progesterone (cat. no. CEA459Ge, 96T) were purchased from Cloud-Clone Corp. (Houston, TX, USA).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Real-time Polymerase Chain Reaction, Transfection, Western Blot, Concentration Assay, Small Interfering RNA, Over Expression

Journal: Nature Communications

Article Title: Harnessing artificial intelligence to identify Bufalin as a molecular glue degrader of estrogen receptor alpha

doi: 10.1038/s41467-025-62288-7

Figure Lengend Snippet: a The entire process of target prediction for Bufalin. b Venn diagram of predicted targets by four web servers and the FMBS method. c Bubble chart of KEGG enrichment analysis for 53 targets. d Chord diagram of pathways with statistically significant p -values and those corresponding to cancer, as retained by KEGG analysis. This diagram illustrates these pathways and their corresponding targets, leading to the selection of 11 key targets. e A multi-task neural network model was developed for these 11 targets, resulting in the identification of 5 high-confidence targets: CYP17A1, ESR1, mTOR, AR, and PRKCD.

Article Snippet: The recombinant Human CYP17A1 (cat. no. CSB-EP006392HU) and estrogen receptor (ESR1) (Cat. no. CSB-YP007830HU) proteins were purchased from CUSABIO .

Techniques: Selection

Journal: Nature Communications

Article Title: Harnessing artificial intelligence to identify Bufalin as a molecular glue degrader of estrogen receptor alpha

doi: 10.1038/s41467-025-62288-7

Figure Lengend Snippet: SPRi graph showing the interaction of Bufalin with ESR1 ( a ), PKC delta ( b ) and CYP17A1 ( c ) recombinant protein. d 293 T cell was transfected with ERα plasmid, after transfected 48 h, the cell lysates were incubated with D-Biotin or Biotin-Bufalin at 4 °C overnight, followed by pulling-down with streptavidin magnetic beads. The proteins bound to the magnetic beads were separated by SDS-PAGE, followed by western blot using ERα antibody. e MCF-7 cell lysates were incubated with D-Biotin or Biotin-Bufalin at 4 °C overnight, followed by pulling-down with streptavidin magnetic beads. The proteins bound to the magnetic beads were separated by SDS-PAGE, followed by western blot using ERα antibody. f 293 T cell was transfected with ERα plasmid, after transfected 48 h, the cell lysates were incubated with a series of concentrations of Biotin-Bufalin at 4 °C overnight, followed by pulling-down with streptavidin magnetic beads. The proteins bound to the magnetic beads were separated by SDS-PAGE, followed by western blot using ERα antibody. g 293 T cell was transfected with Flag-ERα plasmid, after transfected 48 h, the cells were incubated with Bufalin, then the cell lysates were incubated with Biotin-Bufalin at 4 °C, followed by pulling-down with streptavidin magnetic beads. The proteins bound to the magnetic beads were separated by SDS-PAGE, followed by western blot using Flag antibody. h The thermal shift assay experiment (CETSA) was used to evaluate the binding interaction between Bufalin and ERα, the data are presented as mean ± SD, n = 3 independent experiments. i. The cellular location of ERα and Biotin-Bufalin was examined by immunofluorescence staining in MCF-7 cells (Scale bar 5μm). Representative data are shown from n = 3 independent experiments with consistent results.

Article Snippet: The recombinant Human CYP17A1 (cat. no. CSB-EP006392HU) and estrogen receptor (ESR1) (Cat. no. CSB-YP007830HU) proteins were purchased from CUSABIO .

Techniques: Recombinant, Transfection, Plasmid Preparation, Incubation, Magnetic Beads, SDS Page, Western Blot, Thermal Shift Assay, Binding Assay, Immunofluorescence, Staining

Journal: Nature Communications

Article Title: Harnessing artificial intelligence to identify Bufalin as a molecular glue degrader of estrogen receptor alpha

doi: 10.1038/s41467-025-62288-7

Figure Lengend Snippet: a Bufalin was docked into the binding pocket of ESR1 using the Glide SP protocol. b The docked structure of the ESR1-Bufalin complex was then subjected to 100 ns of molecular dynamics simulations using Amber, followed by MM-GBSA energy calculations and residue energy decomposition. The top 10 residues from the MM-GBSA residue energy decomposition scoring. c The top 10 residues selected for alanine scanning mutagenesis. Error values were derived from energy snapshots based on 100 frames uniformly extracted from the last 100 ns of the MD simulation trajectory. d 293 T cell was transfected with ERα wild type (WT) plasmid or mutant plasmid, after transfected 48 h, the cell lysates were incubated with Biotin-Bufalin at 4 °C overnight, followed by pulling-down with streptavidin magnetic beads. The proteins bound to the magnetic beads were separated by SDS-PAGE, followed by western blot using ERα antibody. e 293 T cells were transfected with ERα WT or mutant plasmid, followed by treatment with Bufalin for 48 h. The ERα protein levels were measured by western blot. f 293 T cells were transfected with Flag-ERα WT or R394A mutant plasmid and HA-Ub plasmid, and then subjected to Bufalin for 48 h, followed by treatment with MG-132 (10 μM) for 10 hours before harvest. Then the cells lysates were subjected immunoprecipitation with anti-Flag antibodies and blotted with anti-HA antibodies.

Article Snippet: The recombinant Human CYP17A1 (cat. no. CSB-EP006392HU) and estrogen receptor (ESR1) (Cat. no. CSB-YP007830HU) proteins were purchased from CUSABIO .

Techniques: Binding Assay, Residue, Mutagenesis, Derivative Assay, Transfection, Plasmid Preparation, Incubation, Magnetic Beads, SDS Page, Western Blot, Immunoprecipitation

Journal: Nature Communications

Article Title: Harnessing artificial intelligence to identify Bufalin as a molecular glue degrader of estrogen receptor alpha

doi: 10.1038/s41467-025-62288-7

Figure Lengend Snippet: a The colony formation assay was used to measure MCF-7 and T47D cell proliferation after treatment by Bufalin, the data are presented as mean ± SD, n = 3 independent experiments. One-way ANOVA was used for statistical analysis, P < 0.05 was considered to be statistically significant. b MCF-7 and T47D cells were treated with a series of concentrations of Bufalin, and cell viability was determined using the CCK-8 assay, the data are presented as mean ± SD, n = 3 independent experiments. One-way ANOVA was used for statistical analysis, P < 0.05 was considered to be statistically significant. c MCF-7 cells were treated with 20 or 50 nM Bufalin for 48 h, and the apoptosis was examined by measuring Annexin V staining, the data are presented as mean ± SD, n = 3 independent experiments. One-way ANOVA was used for statistical analysis, P < 0.05 was considered to be statistically significant. d , e MCF-7 cells were transfected with nontargeting siRNA or ESR1 siRNA followed by treatment with Bufalin for 48 h, and apoptosis was examined by measuring Annexin V staining, the data are presented as mean ± SD, n = 3 independent experiments. One-way ANOVA was used for statistical analysis, P < 0.05 was considered to be statistically significant. f MCF-7 cells were transfected with nontargeting siRNA or ESR1 siRNA followed by treatment with Bufalin for 48 h, and the ERα and PARP protein levels were measured by western blot, the samples derive from the same experiment and that gels/blots were processed in parallel. g T47D cells were transfected with nontargeting siRNA or ESR1 siRNA followed by treatment with Bufalin, and the cell viability was determined using the CCK-8 assay, the data are presented as mean ± SD, n = 3 independent experiments. Two-way ANOVA was used for statistical analysis, P < 0.05 was considered to be statistically significant. h 293 T cells were transfected with Flag-ERα plasmid followed by treatment with Bufalin, and the cell viability was determined using the CCK-8 assay, the data are presented as mean ± SD, n = 3 independent experiments. Two-way ANOVA was used for statistical analysis, P < 0.05 was considered statistically significant. i MCF-7 cells were transfected with nontargeting siRNA or ESR1 siRNA followed by treatment with Bufalin, and the colony formation assay was used to measure cell proliferation, the data are presented as mean ± SD, n = 3 independent experiments. One-way ANOVA was used for statistical analysis, P < 0.05 was considered to be statistically significant.

Article Snippet: The recombinant Human CYP17A1 (cat. no. CSB-EP006392HU) and estrogen receptor (ESR1) (Cat. no. CSB-YP007830HU) proteins were purchased from CUSABIO .

Techniques: Colony Assay, CCK-8 Assay, Staining, Transfection, Western Blot, Plasmid Preparation

Journal: Molecular Medicine Reports

Article Title: LKB1 alleviates high glucose- and high fat-induced inflammation and the expression of GnRH and sexual precocity-related genes, in mouse hypothalamic cells by activating the AMPK/FOXO1 signaling pathway

doi: 10.3892/mmr.2022.12659

Figure Lengend Snippet: LKB1 overexpression inhibits GnRH and PP-related protein expression in HG- and HF-induced GT1-7 cells. (A) RT-qPCR was performed to examine GnRH expression. (B-C) Measurement of ERβ, CD36 and GPR54 expression using western blot analysis. **P<0.01 and ***P<0.001. LKB1, live kinase B1; GnRH, gonadotropin-releasing hormone; PP, precocious puberty; HF, high fat; HG, high glucose; RT-qPCR, reverse transcription-quantitative qPCR; ERβ, estrogen receptor-β; CD36, cluster of differentiation 36; GPR54, G-protein-coupled receptor; Oe, overexpression; NC, negative control.

Article Snippet: Anti-LKB1 (cat. no. 13031T; 1:1,000), anti-IL-6 (cat. no. 12912T; 1:1,000), anti-TNF-α (cat. no. 11948T; 1:1,000), anti-CD36 (cat. no. 74002S; 1:1,000), anti-phosphorylated (p)-AMPK (cat. no. 2535T; 1:1,000), anti-AMPK (cat. no. 5831T; 1:1,000), anti-p-FOXO1 (cat. no. 84192S; 1:1,000), anti-FOXO1 (cat. no. 2880T; 1:1,000) and anti-GAPDH (cat. no. 5174S; 1:1,000) antibodies were obtained from Cell Signaling Technology, Inc. Anti-estrogen receptor-β (ERβ; ab196787; 1:1,000) and anti-G-protein-coupled receptor (GPR54; cat. no. ab100896: 1:1,000) antibodies were provided by Abcam.

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Western Blot, Negative Control

Journal: Molecular Medicine Reports

Article Title: LKB1 alleviates high glucose- and high fat-induced inflammation and the expression of GnRH and sexual precocity-related genes, in mouse hypothalamic cells by activating the AMPK/FOXO1 signaling pathway

doi: 10.3892/mmr.2022.12659

Figure Lengend Snippet: Inactivation of AMPK/FOXO1 signaling reversed the impacts of LKB1 overexpression on inflammation and GnRH expression in HG- and HF-induced GT1-7 cells. Detection of LKB1 (A) mRNA and (B) protein expression using RT-qPCR and western blot analysis. (C) RT-qPCR was performed to examine GnRH expression. (D) Measurement of ERβ, CD36 and GPR54 expression using western blot analysis. **P<0.01 and ***P<0.001. AMPK, AMP-activated protein kinase; FOXO1, forkhead box protein O1; LKB1, live kinase B1; GnRH, gonadotropin-releasing hormone; HF, high fat; HG, high glucose; RT-qPCR, reverse transcription-quantitative qPCR; ERβ, estrogen receptor-β; CD36, cluster of differentiation 36; GPR54, G-protein-coupled receptor; Oe, overexpression; NC, negative control.

Article Snippet: Anti-LKB1 (cat. no. 13031T; 1:1,000), anti-IL-6 (cat. no. 12912T; 1:1,000), anti-TNF-α (cat. no. 11948T; 1:1,000), anti-CD36 (cat. no. 74002S; 1:1,000), anti-phosphorylated (p)-AMPK (cat. no. 2535T; 1:1,000), anti-AMPK (cat. no. 5831T; 1:1,000), anti-p-FOXO1 (cat. no. 84192S; 1:1,000), anti-FOXO1 (cat. no. 2880T; 1:1,000) and anti-GAPDH (cat. no. 5174S; 1:1,000) antibodies were obtained from Cell Signaling Technology, Inc. Anti-estrogen receptor-β (ERβ; ab196787; 1:1,000) and anti-G-protein-coupled receptor (GPR54; cat. no. ab100896: 1:1,000) antibodies were provided by Abcam.

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Western Blot, Negative Control

Journal: Poultry science

Article Title: Synergistic effect of genistein and adiponectin reduces fat deposition in chicken hepatocytes by activating the ERβ-mediated SIRT1-AMPK signaling pathway.

doi: 10.1016/j.psj.2024.103734

Figure Lengend Snippet: Figure 3. Genistein promotes adiponectin secretion through binding with ERb in adipocytes. ICP-1 cells were pretreated with vehicle, GPER1 inhibitor G15 (1 mM; 12h), GPER1 activator G1 (1 mM; 1h), ER antagonist Fulvestrant (1 mM; 12h) or ERb antagonist PHTPP (10 mM; 1h); and then the cells were treated with 0 or 40 mM GEN for another 24 h. (A) Immunoblot of p-Erk, t-Erk, PPARg, and Tublin b protein; (B−C) The pro- tein levels of p-Erk/t-Erk and PPARg; (D) APN mRNA expression; (E) Immunoblot of p-Erk, t-Erk, PPARg, and Tublin b protein; (F−G) The protein levels of p-Erk/t-Erk and PPARg; (H) APN mRNA expression. (I) Immunoblot of p-Erk, t-Erk, PPARg, and Tublin b protein; (J−K) The protein levels of p-Erk/t-Erk and PPARg; L: APN mRNA expression; Data are expressed as means § SEM (n = 3). ** P < 0.01, comparison with the respective control groups; NS, no significant difference between the indicated groups.

Article Snippet: ERb-Erk-PPARg Signaling Pathway Analysis The ICP-1 cells were seeded in 6-well plates and preincubated with 1 mM GPER1 inhibitor G15 (Cat#S6651, Selleck, Houston, TX) for 12 h, 1 mM GPER1 activator G1 (Cat#S0851, Selleck) for 1 h, 1 mM estrogen receptor antagonist Fulvestrant (Cat#S1191, Selleck) for 12 h, 10 mM ERb antagonist PHTPP (Cat#S8686, Selleck) for 1 h, 10 mMErk inhibitor U0126 (Cat#S1102, Selleck) for 4 h or 5 mM PPARg inhibitor GW9662 (Cat#S2915, Selleck) for 4 h, respectively.

Techniques: Binding Assay, Western Blot, Expressing, Comparison, Control