Journal: The Journal of steroid biochemistry and molecular biology

Article Title: Effect of sildenafil on human aromatase activity: From in vitro structural analysis to catalysis and inhibition in cells.

doi: 10.1016/j.jsbmb.2016.09.003

Figure Lengend Snippet: Figure 1. UV-vis spectroscopy shows a Type II binding of sildenafil to human aromatase. A) Visible direct

Article Snippet: Confluent adherent cells grown in 10 cm diameter dishes were transiently transfected in Opti-MEM reduced serum medium (Gibco) by mixing 10 μg of the expression vector pCMV6-XL4 carrying the cDNA coding for the full-length aromatase (Origene, Rockville, USA) or a control empty vector (pIRES-puro2) and 10 μL of Lipofectamine2000 (Invitrogen) according to the manufacturer recommendations.

Techniques: UV-Vis Spectroscopy, Binding Assay

Journal: Cerebral cortex (New York, N.Y. : 1991)

Article Title: Roles of 17ß-estradiol involve regulation of reelin expression and synaptogenesis in the dentate gyrus.

doi: 10.1093/cercor/bhq047

Figure Lengend Snippet: Figure 3. Expression of aromatase in DG. (A) Distribution of aromatase in DG of an early postnatal (P10) rat as determined by immunostaining using a polyclonal antiserum derived from rabbit (Dr Azcoitia, Madrid). Note: Immunoreactivity was robust in the outer part of the GCL. In contrast, little signal was detected in the inner part of GCL, which contains immature granule cells (Bender et al. 2001). Strong immunoreactivity for aromatase was also found in hilar (hil) neurons. (B) In mature DG (P60), aromatase immunoreactivity is more diffusely distributed and appears to mainly localize to granule cell dendrites. (C) A similar staining pattern was found with a different, monoclonal anti- aromatase antibody (Acris). Colocalization of aromatase with NeuN, a marker for mature neurons, further confirmed that the enzyme is mainly expressed by mature granule cells in immature DG (D, E). Scale bar: 25 lm (A), 20 lm (B--E).

Article Snippet: For IHC, both brain and culture sections were preincubated with 3% normal goat serum (in PBS) for 1 h at room temperature (RT) and then primary antibodies were applied for 48 h at 4 C. The following antibodies were used: polyclonal rabbit-anti-ERa C-terminus (Santa Cruz Biotechnology, HC-20, directed against the C-terminus of human ERa; 1:100), polyclonal rabbit-anti-ERa N-terminus (Santa Cruz, H-184, directed against AA 2-185 of human ERa; 1:100), polyclonal rabbit-anti-ERb (Dianova, PAT-311, directed against AA 376-388 of mouse ERb; 1:100; antibody showed distinct immunolabeling in paraventricular nucleus of hypothalamus, where ERb is robustly expressed; Orikasa et al. 2000; data not shown), polyclonal goat-antiNotch1 (Santa Cruz, sc-6015, 1:500; directed against the C-terminal fragment of Notch1), polyclonal rabbit-anti-aromatase (Yague et al. 2006; directed against AA 488-503 of mouse aromatase; 1:1200; kind gift of Dr I. Azcoitia, Madrid), monoclonal mouse-anti-aromatase (Acris Antibodies, SM2222P; directed against AA 376-390 of human aromatase; 1:60), monoclonal mouse-anti-reelin (Millipore, 1:500), and monoclonal mouse-anti-NeuN (Millipore, 1:200).

Techniques: Expressing, Immunostaining, Derivative Assay, Staining, Marker

Journal: International Journal of Molecular Sciences

Article Title: Immunofluorescent Evidence for Nuclear Localization of Aromatase in Astrocytes in the Rat Central Nervous System

doi: 10.3390/ijms23168946

Figure Lengend Snippet: List of primary antibodies used in this study. According to the technical information provided by the manufacturers, the commercial primary antibodies used in the study were verified by Knockdown or Relative expression to ensure that the antibody binds to the antigen stated.

Article Snippet: Aromatase NB100-1596 (Novus Biologicals) AB_10000919 , Rabbit/Polyclonal , C-terminal portion of the human aromatase protein (between residues 400–502) , Human, Mouse, Rat, Primate, Bovine, Rabbit , 1:100 , [ ] .

Techniques: Knockdown, Expressing, Binding Assay, Recombinant

Journal: Frontiers in Endocrinology

Article Title: The interplay between kisspeptin and endocannabinoid systems modulates male hypothalamic and gonadic control of reproduction in vivo

doi: 10.3389/fendo.2023.1269334

Figure Lengend Snippet: Primary and secondary antisera used for Western blot, IFL, and IHC.

Article Snippet: Aromatase (Cyp19) anti-rabbit polyclonal IgG (E-AB-64300, Elabscience: Houston, TX, USA) , 1:2000.

Techniques: Western Blot, Plasmid Preparation

Journal: Frontiers in Endocrinology

Article Title: The interplay between kisspeptin and endocannabinoid systems modulates male hypothalamic and gonadic control of reproduction in vivo

doi: 10.3389/fendo.2023.1269334

Figure Lengend Snippet: Modulation of Kiss1R, Kiss1, Cyp19, GnRH , and miR expression in mediobasal hypothalamus homogenates. Representative Western blots ( n = 4) for Kiss1R, Kiss1, and Cyp19 (A) and normalization of Kiss1R (B) , Kiss1 (C) , and Cyp19 signals (D) carried out against α-tubulin. Quantitative expression ( n = 6) of GnRH (E) and miR-132-3p , Mir-145-5p , let-7a-5p , and let-7b-5p (F–I) carried out by qPCR. Data are expressed as protein OD/tubulin OD ± standard deviation or normalized fold expression (n.f.e.) ± SEM against the housekeeping genes HPRT / β-actin / U6 and the control group used as reference sample. C, control group (placebo injected animals); KP, animals injected with Kp10; AEA, animals injected with AEA; SR + AEA (SA), animals first received SR141716A and 30 min later received AEA treatment. ** p < 0.01, * p < 0.05 vs. the control group.

Article Snippet: Aromatase (Cyp19) anti-rabbit polyclonal IgG (E-AB-64300, Elabscience: Houston, TX, USA) , 1:2000.

Techniques: Expressing, Western Blot, Standard Deviation, Control, Injection

Journal: Frontiers in Endocrinology

Article Title: The interplay between kisspeptin and endocannabinoid systems modulates male hypothalamic and gonadic control of reproduction in vivo

doi: 10.3389/fendo.2023.1269334

Figure Lengend Snippet: Modulation of the intratesticular markers of steroidogenesis and GnRH . Representative Western blots for 3βHsd and Cyp19 (A) and normalization of 3βHsd (B) and Cyp19 (C) signals carried out against α-tubulin. Quantitative expression of 3βHsd , Cyp19 , and GnRH mRNA carried out by qPCR (D) . Data are representative of n = 6 different animals and are expressed as protein OD/tubulin OD ± standard deviation or normalized fold expression (n.f.e.) ± SEM against the housekeeping genes HPRT / β-actin and the control group used as a reference sample. C, control group (placebo-injected animals); KP, animals injected with Kp10; * p < 0.05.

Article Snippet: Aromatase (Cyp19) anti-rabbit polyclonal IgG (E-AB-64300, Elabscience: Houston, TX, USA) , 1:2000.

Techniques: Western Blot, Expressing, Standard Deviation, Control, Injection

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: A novel variant of ER-alpha, ER-alpha36 mediates testosterone-stimulated ERK and Akt activation in endometrial cancer Hec1A cells

doi: 10.1186/1477-7827-7-102

Figure Lengend Snippet: Letrozole inhibits ER-α36-mediated ERK and Akt phosphorylation . Hec1A cells were pre-treated with 10 nM aromatase inhibitor letrozole (Lanes 3 and 4) for 1 h and then the cells were treated with vehicle (DMSO) (Lanes 1) and 10 nM testosterone (Lanes 2 and 4) for 5 min. Phospho-ERK1/2 (A) or phospho-Akt (B) were examined with specific antibodies. The same blot was stripped and probed with anti-total ERK or Akt antibodies. Hec1A cells were treated with 10 nM testosterone (Lanes 2) or together with 10 nM aromatase inhibitor letrozole (Lanes 3) overnight, and then aromatase expression was detected by Western blot using specific antibody against aromatase (C).

Article Snippet: Anti-aromatase antibody was purchased from Novus Biologicals (Novus Biologicals, Littleton, CO).

Techniques: Phospho-proteomics, Expressing, Western Blot

Journal: Journal of Virology

Article Title: Testicular Degeneration and Infertility following Arbovirus Infection

doi: 10.1128/JVI.01131-18

Figure Lengend Snippet: 3β-HSD and P450 expression in BTV-1 experimentally infected rams and mock-infected controls. (A and B) Immunohistochemistry showing expression of 3β-HSD (A) and P450 (B) (in brown) by the Leydig cells in sections of the testes of mock-infected healthy control rams. Arrows point to Leydig cells. (C to F) Immunohistochemistry showing lack of expression of 3β-HSD (C and E) and P450 (D and F) by Leydig cells in the testes of rams infected with BTV-1 IT2006 (C and D) or BTV-1 IT2013 (E and F). The peritubular areas where the Leydig cells are located are highlighted with a broken line. Scale bars, 100 μm. (G) Graph representing the relative levels of testosterone, assessed by ELISA, in the blood of rams infected with either BTV-1 IT2006 or BTV-1 IT2013 . Values are shown as percentages of the values of testosterone taken at day 0 before virus infection. Note the significant reduction in the levels of testosterone in rams infected with BTV-1 IT2006 between 0 and 8 to 11 dpi ( P < 0.05; one-way ANOVA) and in rams infected with BTV-1 IT2013 between 0 and either 5 or 15 dpi ( P < 0.05; one-way ANOVA).

Article Snippet: Sections were incubated overnight at 4°C using primary antibodies specific against the following markers: BTV NS2 ( , ), vimentin (Dako Agilent), von Willebrand factor (Dako Agilent), CD3 (Dako Agilent), MX-1 ( , ), 3β-HSD (Santa Cruz Biotechnology), P450 Aromatase (AcrisAntibodies GmbH), inhibin α (Ventana Medical Systems), smooth muscle actin (Dako Agilent), and KI-67 (Dako Agilent).

Techniques: Expressing, Infection, Immunohistochemistry, Control, Enzyme-linked Immunosorbent Assay, Virus

Journal: Nature genetics

Article Title: Acquired CYP19A1 amplification is an early specific mechanism of aromatase inhibitor resistance in ERα metastatic breast cancer.

doi: 10.1038/ng.3773

Figure Lengend Snippet: Figure 3 CYP19A1 amplification leads to increased aromatase activity. (a) CNA profiles for the CYP19A1 and ESR1 loci in treatment-naive (MCF7) and estrogen-deprived (LTED) cells. Boxes and error bars, mean ± s.d. of 3 technical replicates. Data are normalized to the TERT locus. (b) DNA- FISH using CYP19A1-centered probes identifies widespread cluster amplification in LTED cells. (c,d) RT-qPCR (c) and immunoblot (d) analysis of aromatase expression at the CYP19A1 locus in LTED clones. T suffix, tamoxifen-resistant; F, fulvestrant-resistant. Data represent mean ± s.e.m. from 3 independent experiments (full blots shown in Supplementary Fig. 11). (e) Single-cell RNA-FISH highlights heterogeneity in aromatase expression. ****P < 10−20, Mann-Whitney test. (f) Transcriptional activation of estrogen target genes in CYP19A1amp cells treated with ethanol (mock) or aromatizable androstenedione (and.) alone or in combination with letrozole (let.). Data are mean ± s.e.m. from 5 independent experiments. *P < 0.05, two-way or one-way ANOVA (bottom right).

Article Snippet: CYP19A1-overexpressing cells were obtained by transfecting MCF7 cells with full-length CYP19A1 (RC205890, OriGene Technologies) and selection using G418.

Techniques: Amplification, Activity Assay, Quantitative RT-PCR, Western Blot, Expressing, Clone Assay, MANN-WHITNEY, Activation Assay

Journal: Nature genetics

Article Title: Acquired CYP19A1 amplification is an early specific mechanism of aromatase inhibitor resistance in ERα metastatic breast cancer.

doi: 10.1038/ng.3773

Figure Lengend Snippet: Figure 4 CYP19A1amp cells endogenously activate ERα and develop tolerance to AI. (a) ChIP-seq heat maps for ERα in MCF7 and LTED cells. Binding sites have been assigned to three clusters. The average profile of each cluster is shown (middle). Examples of ERα enrichment near important estrogen target genes are shown in the insets (right). (b) IC50 growth curve for MCF7 cell treated with increasing doses of AI in combination with estradiol. (c) LTED treatment with AI in the absence of estradiol. (d) LTED cells treated with siRNA against CYP19A1 (siCYP19A1-1 and siCYP19A1-2) have increased sensitivity to AI. SRB, sulforhodamine B assay; siC, control siRNA targeting luciferase. (e) CYP19A1-overexpressing cells have a growth advantage over wild-type in the absence of estradiol. Relative increase in growth rate is shown as the ratio of the growth of CYP19A1-overexpressing cells to CYP19A1 wild-type cells under letrozole challenge. (f) CYP19A1amp LTED cells respond to low levels of irreversible steroidal AI. (g) Kaplan– Meier curve showing time to first relapse (TTFR) for AI-treated patients stratified retrospectively for CYP19A1 amplification. Dotted lines represent 95% CI. HR, hazard ratio. P value determined by log–rank test. (h) Working hypothesis for therapy-specific breast cancer progression. Genetic and epigenetic changes collaborate to increase tumor fitness by creating an estrogen-independent niche at metastatic sites treated with AI therapy. For b–f, data are mean ± s.e.m. (b,d,e) or mean ± s.d. (c) from 3 independent experiments or mean and 95% CI from 4 independent replicates (f). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, Student’s t-test (c) or two-way ANOVA and Bonferroni’s post-test (d–f). A.U., arbitrary units.

Article Snippet: CYP19A1-overexpressing cells were obtained by transfecting MCF7 cells with full-length CYP19A1 (RC205890, OriGene Technologies) and selection using G418.

Techniques: ChIP-sequencing, Binding Assay, Sulforhodamine B Assay, Control, Luciferase, Amplification