esr2 antagonist  (MedChemExpress)

Journal: Current Issues in Molecular Biology

Article Title: Biphasic Slc2a4 Gene Expression in 3T3-L1 Adipocytes in Response to Treatment with Low and High Concentrations of Daidzein and Genistein

doi: 10.3390/cimb47100857

Figure Lengend Snippet: The enhancer effect of low concentrations of daidzein and genistein on the Slc2a4 gene expression in adipocytes involves the activation of estrogen receptor 1 (ESR1). Slc2a4 mRNA expression was analyzed in differentiated 3T3-L1 adipocytes cultivated, for 24 h, with 17β-estradiol ( A , B ), daidzein ( C , D ) and genistein ( E , F ), at equimolar concentrations (10 nM), combined or not with 1 µM ESR1 antagonist MPP (1,3-Bis(4-hydroxyphenyl)-4-methyl-5-[4-(piperidinylethoxy)phenol]-1H-pyrazol dihydrochloride) ( A , C , E ) or 1 µM ESR2 antagonist PHTPP (4-[2-Phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol) ( B , D , F ), and compared with a control treatment without hormone. Slc2a4 mRNA was analyzed by RT-qPCR, using the Gapdh mRNA as a loading control. Data are expressed as mean ± SD of 10 (E2P group), 8 (C groups), 6 or 7 (the other groups) samples, from at least 3 different batches of experiments. The means were compared by one-way ANOVA, followed by Tukey’s multiple comparison test (* p < 0.05 vs. C, ** p < 0.01, *** p < 0.001 vs. C; ### p < 0.001 vs. E2; & p < 0.05 vs. M; §§§ p < 0.001 vs. D; £££ p < 0.001 vs. G). C, control; E2, 17β-estradiol; D, daidzein; G, genistein; M, ESR1 antagonist MPP; P, ESR2 antagonist PHTPP; AU, arbitrary units; Slc2a4 , solute carrier family 2 member 4; Gapdh , glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: On day 10, cells were transferred to DMEM with 5.5 mM glucose, 10% FBS, 1% antibiotics (penicillin/streptomycin) (Vitrocell Embriolife, Campinas, SP, Brazil), and 1.5 nM insulin ((Sigma-Aldrich; St. Louis, MO, USA); thus, 24 h treatments were performed with 10 nM of water-soluble 17β-estradiol (E2) (E4389, Sigma-Aldrich, St. Louis, MO, USA); 10 nM, 50 μM or 150 μM daidzein (D) (7802, Sigma-Aldrich, St. Louis, MO, USA); 10 nM, 5 μM or 50 μM genistein (G) (G6649, Sigma-Aldrich, St. Louis, MO, USA), added or not with 1 μM of a selective ESR1 antagonist MPP (M) [1,3-bis(4-hydroxyphenyl)-4-methyl-5-(4-(2-piperidinylethoxy)phenol)-1H-pyrazole dihydrochloride; Tocris, Avonmouth, Bristol, UK]; or 1 μM of a selective ESR2 antagonist PHTPP (P) [(2-Phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl)phenol; Tocris, Avonmouth, Bristol, UK].

Techniques: Gene Expression, Activation Assay, Expressing, Control, Quantitative RT-PCR, Comparison

Journal: Current Issues in Molecular Biology

Article Title: Biphasic Slc2a4 Gene Expression in 3T3-L1 Adipocytes in Response to Treatment with Low and High Concentrations of Daidzein and Genistein

doi: 10.3390/cimb47100857

Figure Lengend Snippet: The repressor effect of high concentrations of daidzein and genistein on Slc2a4 gene expression in adipocytes seems to involve ESR1 inhibition and ESR2 activation. The Slc2a4 mRNA expression was analyzed in differentiated 3T3-L1 adipocytes cultivated, for 24 h, with 150 µM daidzein ( A , C ) and 50 µM genistein ( B , D ), combined or not with 1 µM ESR1 antagonist MPP (1,3-Bis(4-hydroxyphenyl)-4-methyl-5-[4-(piperidinylethoxy)phenol]-1H-pyrazol dihydrochloride) ( A , B ) or 1 µM ESR2 antagonist PHTPP (4-[2-Phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol) ( C , D ), and compared with a control treatment without hormone. The Slc2a4 mRNA was analyzed by RT-qPCR, using the Gapdh mRNA as a loading control. Data are expressed as mean ± SD of 6 samples, from at least 3 different batches of experiments. The means were compared by one-way ANOVA, followed by Tukey’s multiple comparison test (** p < 0.01, *** p < 0.001 vs. C; §§ p < 0.01 vs. D; £ p < 0.05 vs. G; &&& p < 0.001 vs. M; ¶¶¶ p < 0.001 vs. P). C, control; D, daidzein; G, genistein; M, ESR1 antagonist MPP; P, ESR2 antagonist PHTPP; AU, arbitrary units; Slc2a4 , solute carrier family 2 member 4; Gapdh , glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: On day 10, cells were transferred to DMEM with 5.5 mM glucose, 10% FBS, 1% antibiotics (penicillin/streptomycin) (Vitrocell Embriolife, Campinas, SP, Brazil), and 1.5 nM insulin ((Sigma-Aldrich; St. Louis, MO, USA); thus, 24 h treatments were performed with 10 nM of water-soluble 17β-estradiol (E2) (E4389, Sigma-Aldrich, St. Louis, MO, USA); 10 nM, 50 μM or 150 μM daidzein (D) (7802, Sigma-Aldrich, St. Louis, MO, USA); 10 nM, 5 μM or 50 μM genistein (G) (G6649, Sigma-Aldrich, St. Louis, MO, USA), added or not with 1 μM of a selective ESR1 antagonist MPP (M) [1,3-bis(4-hydroxyphenyl)-4-methyl-5-(4-(2-piperidinylethoxy)phenol)-1H-pyrazole dihydrochloride; Tocris, Avonmouth, Bristol, UK]; or 1 μM of a selective ESR2 antagonist PHTPP (P) [(2-Phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl)phenol; Tocris, Avonmouth, Bristol, UK].

Techniques: Gene Expression, Inhibition, Activation Assay, Expressing, Control, Quantitative RT-PCR, Comparison

Journal: bioRxiv

Article Title: A gatekeeping role of ESR2 to maintain the primordial follicle reserve

doi: 10.1101/2020.02.06.937953

Figure Lengend Snippet: Gonadotropin stimulation resulted in an increased number of antral follicles in Esr2-/- ovaries. Wildtype (WT) and Esr2-/- rats were treated with exogenous gonadotropins on PND28. 48h after PMSG (30IU) injection, rats were administered with hCG (30IU). 4h after the hCG injection, ovaries were collected and processed for histological examination or collection of COCs. Histological examination demonstrated the presence of ovarian follicles at different stages of development in WT rats ( A ). In contrast, Esr2-/- rat ovaries showed an increased number of antral follicles ( B ). Cumulus cells were detached from the oocytes by mechanical pipetting before counting under microscope. Oocyte yield was about three-fold higher in Esr2-/- rat ovaries ( C ). Serially sectioned whole ovaries were stained with H&E, and follicles at different stages were counted in WT and Esr2-/- rats ( D ). Follicle counting demonstrated a decreased number of primordial follicles and an increased number of activated follicles in Esr2-/- rats ( E-G ). Data shown as mean ± SE, n≥3. * P ≤ 0.05.

Article Snippet: A selective ESR2 antagonist PHTPP ( ) or a selective ESR2 agonist DPN ( ) (Cayman Chemical, Ann Arbor, MI) was dissolved in DMSO (5µg/µl).

Techniques: Injection, Microscopy, Staining

Journal: bioRxiv

Article Title: A gatekeeping role of ESR2 to maintain the primordial follicle reserve

doi: 10.1101/2020.02.06.937953

Figure Lengend Snippet: Increased activation of primordial follicles in Esr2-/- rat ovaries. Follicle counting in PND28 ( A-C, J, M ), PND16 ( D-F, K, N ), and PND8 ( G-I, L, O ) wildtype (WT) and Esr2-/- rats showed increased activation of primordial follicles in Esr2-/- ovaries. Primordial follicle activation was near two-fold starting on PND8 ( G-I, L, O ). A greater number of atretic follicles were present within the pool of activated follicles at PND28 ( A, B ) PND16 ( D, E ) and PND8 ( G, H ) in Esr2-/- ovaries. Data shown as mean ± SE, n≥3. * P ≤ 0.05. Pd, Primordial follicle; Py, Primary follicle.

Article Snippet: A selective ESR2 antagonist PHTPP ( ) or a selective ESR2 agonist DPN ( ) (Cayman Chemical, Ann Arbor, MI) was dissolved in DMSO (5µg/µl).

Techniques: Activation Assay

Journal: bioRxiv

Article Title: A gatekeeping role of ESR2 to maintain the primordial follicle reserve

doi: 10.1101/2020.02.06.937953

Figure Lengend Snippet: Premature ovarian senescence in Esr2-/- rats. The total number of follicles in PND8 ovaries were similar between wildtype (WT) and Esr2-/- rats; however, the number of primordial follicles were decreased in Esr2-/- ovaries ( A ). Follicle counts in 4wk, 12wk, and 24wk old WT and Esr2-/- rats revealed a sharp decline in the primordial follicle reserve in Esr2-/- ovaries ( B-H ). 24wk old Esr2-/- rats showed a significantly lower level of serum estradiol and AMH compared to WT ( I, J ). Data shown as mean± SE, n≥3 (follicle counting) and n≥6 (hormone assays), * P ≤ 0.05. Pd, Primordial follicle; Py, Primary follicle.

Article Snippet: A selective ESR2 antagonist PHTPP ( ) or a selective ESR2 agonist DPN ( ) (Cayman Chemical, Ann Arbor, MI) was dissolved in DMSO (5µg/µl).

Techniques:

Journal: bioRxiv

Article Title: A gatekeeping role of ESR2 to maintain the primordial follicle reserve

doi: 10.1101/2020.02.06.937953

Figure Lengend Snippet: Regulation of primordial follicle activation is ESR2-dependent. Deletion of the ESR2 DBD increased primordial follicle activation at PND28 ovaries ( A, B, G, I, J ), but this was not observed in ovaries from Esr1-/- rats ( C, G, K ). Administration of a selective ESR2 antagonist, PHTPP, into wildtype rats increased primordial follicle activation ( D, E, H, L, M ), whereas treatment with an ESR2 agonist, DPN, suppressed the activation ( F, H, N ). Data shown as mean ± SE, n ≥ 3. * P ≤ 0.05. Rel., Relative. Pd, Primordial follicle; Py, Primary follicle.

Article Snippet: A selective ESR2 antagonist PHTPP ( ) or a selective ESR2 agonist DPN ( ) (Cayman Chemical, Ann Arbor, MI) was dissolved in DMSO (5µg/µl).

Techniques: Activation Assay

Journal: bioRxiv

Article Title: A gatekeeping role of ESR2 to maintain the primordial follicle reserve

doi: 10.1101/2020.02.06.937953

Figure Lengend Snippet: Activation of AKT, ERK, and mTOR signaling in Esr2-/- ovaries. Expression of ESR2 was detected in PND4, 6, and 8 rat ovaries using RT-qPCR ( A ) and western blotting ( B ). Primordial (Pd) ( C ), and primary (Py) ( D ) follicles were isolated from rat ovaries by digestion with liberase followed by size fractionation with strainers. RT-qPCR analysis demonstrated Esr2 expression in both Pd and Py follicles ( E ). Western blot analyses of PND8 ovaries and quantification of signal intensities demonstrated a significant increase in AKT ( F-I ) and ERK1/2 ( L-M ) activation. This was associated with increased activation of mTORC1 ( P-Q ) and its targets P70S6K ( R-S ) and RPS6 ( T-U ). But no difference was observed in PTEN ( J-K ) and pTSC2 ( N-O ) levels. Signal quantification data are presented as mean ± SEM. n ≥ 6. * P ≤ 0.05. Rel., Relative.

Article Snippet: A selective ESR2 antagonist PHTPP ( ) or a selective ESR2 agonist DPN ( ) (Cayman Chemical, Ann Arbor, MI) was dissolved in DMSO (5µg/µl).

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Western Blot, Isolation, Fractionation

Journal: bioRxiv

Article Title: A gatekeeping role of ESR2 to maintain the primordial follicle reserve

doi: 10.1101/2020.02.06.937953

Figure Lengend Snippet: Transcript levels of known activators of the AKT and mTOR pathways. RT-qPCR was performed on PND8 wildtype (WT) and Esr2-/- rat ovaries to analyze the expression of genes involved in activation of the AKT and mTOR pathways and activation of primordial follicles. While the expression of Kitlg ( A ), Kit ( B ), Igf1 ( C ), Bmp15 ( H ) and Gdf9 ( I ) was moderately upregulated in Esr2-/- rat ovaries, Adcyap1r1 ( F ), Bmp4 ( G ), Gata4 ( N ), and Npm2 ( O ) were highly upregulated. But no significant differences in the expression of Fgf2 ( D ), Ntrk2 ( E ), Amh ( J ), Bdnf ( K ), Nobox ( L ) and Sohlh1 ( M ) were observed between the WT and Esr2-/- rat ovaries. RT-qPCR data represent the mean ± SEM. n ≥ 8. * P ≤ 0.05. Rel., Relative.

Article Snippet: A selective ESR2 antagonist PHTPP ( ) or a selective ESR2 agonist DPN ( ) (Cayman Chemical, Ann Arbor, MI) was dissolved in DMSO (5µg/µl).

Techniques: Quantitative RT-PCR, Expressing, Activation Assay

Journal: bioRxiv

Article Title: A gatekeeping role of ESR2 to maintain the primordial follicle reserve

doi: 10.1101/2020.02.06.937953

Figure Lengend Snippet: ESR2 signaling in the regulation of primordial follicle activation. Loss of ESR2 leads to upregulation of both granulosa cell and oocyte derived factors that can activate the AKT, ERK and mTOR pathways. Increased levels of KITLG, KIT and IGF1, as well as NPM2, and ADCYAP1R1 can activate the AKT pathway followed by the mTOR pathway. Moreover, upregulation of NPM2 and BMP4 can activate the ERK pathway. Activated AKT, ERK and mTOR pathways in association with the transcriptional regulator GATA4 can promote the transition of primordial follicles to primary follicles in Esr2-/- ovaries.

Article Snippet: A selective ESR2 antagonist PHTPP ( ) or a selective ESR2 agonist DPN ( ) (Cayman Chemical, Ann Arbor, MI) was dissolved in DMSO (5µg/µl).

Techniques: Activation Assay, Derivative Assay

Journal: Scientific Reports

Article Title: Estrogen receptor 1 (ESR1) regulates VEGFA in adipose tissue

doi: 10.1038/s41598-017-16686-7

Figure Lengend Snippet: ( a ) mRNA expression of Esr1 and Esr2 after treatment with 10 nM of E2 for 24 hours in 3T3-L1 cells. ( b ) ESR1 total protein expression after treatment with 10 nM of E2 for 24 hours in 3T3-L1 cells. Full-length blots of the cropped image are presented in Supplementary Figure . Control cells were treated with vehicle (water). Data on the graphs are mean ± SEM of 3 to 5 samples. & P < 0.05 vs control.

Article Snippet: The concentrations of E2, ESR1 and ESR2 agonists and antagonists were established according to previous reports , , – : we used 10 nM of the ESR1 agonist, PPT: 4,4′,4′′-(4-Propyl-[1 H ]-pyrazole-1,3,5-triyl) tris phenol (Tocris, St. Louis, MO,USA,; ESR1 antagonist, MPP: 1,3-Bis(4-hydroxyphenyl)-4-methyl-5-[4-(piperidinylethoxy)phenol]-1H-pyrazol dihydrochloride (Tocris) (1μmol/L); ESR2 agonist, DPN: 2,3-bis(4-Hydroxyphenyl)-propionitrile) (Tocris) (100 nM); and 100 nM was used of the ESR2 antagonist: PHTPP, 4-[2-Phenyl-5,7-bis(trifluoromethyl) pyrazolo[1,5-a]pyrimidin-3-yl]phenol (Tocris).

Techniques: Expressing, Control

Journal: Scientific Reports

Article Title: Estrogen receptor 1 (ESR1) regulates VEGFA in adipose tissue

doi: 10.1038/s41598-017-16686-7

Figure Lengend Snippet: Effects of ESR1 agonist (PPT) and ESR2 agonist (DPN) on VEGFA mRNA and protein: 3T3-L1 adipocytes were treated for 24 hours with 10 nM of PPT ( a , b ) or 10 µmol/L of DPN ( c , d ). Full-length blots of these cropped images are presented in Supplementary Figure . Cells treated with vehicle (0.1% DMSO) were used as control. The representative autoradiograms are shown on top. Data are shown as the mean ± SEM of 6 samples. & P < 0.05 vs control.

Article Snippet: The concentrations of E2, ESR1 and ESR2 agonists and antagonists were established according to previous reports , , – : we used 10 nM of the ESR1 agonist, PPT: 4,4′,4′′-(4-Propyl-[1 H ]-pyrazole-1,3,5-triyl) tris phenol (Tocris, St. Louis, MO,USA,; ESR1 antagonist, MPP: 1,3-Bis(4-hydroxyphenyl)-4-methyl-5-[4-(piperidinylethoxy)phenol]-1H-pyrazol dihydrochloride (Tocris) (1μmol/L); ESR2 agonist, DPN: 2,3-bis(4-Hydroxyphenyl)-propionitrile) (Tocris) (100 nM); and 100 nM was used of the ESR2 antagonist: PHTPP, 4-[2-Phenyl-5,7-bis(trifluoromethyl) pyrazolo[1,5-a]pyrimidin-3-yl]phenol (Tocris).

Techniques: Control

Journal: Scientific Reports

Article Title: Estrogen receptor 1 (ESR1) regulates VEGFA in adipose tissue

doi: 10.1038/s41598-017-16686-7

Figure Lengend Snippet: Effects of ESR1 antagonist (MPP) and ESR2 antagonist (PHTPP) on VEGFA mRNA and protein: 3T3-L1 adipocytes were treated for 24 hours with 10 µmol/L of MPP ( a , b ) or 100 nmol/L of PHTPP ( c , d ), in the absence or presence of 10 nM of E2. Full-length blots of these cropped images are presented in Supplementary Figure . Cells treated with vehicle (0.1% DMSO) were used as control. The representative autoradiograms are shown on top. Data are shown as the mean ± SEM of 6 samples. & P < 0.05 vs control.

Article Snippet: The concentrations of E2, ESR1 and ESR2 agonists and antagonists were established according to previous reports , , – : we used 10 nM of the ESR1 agonist, PPT: 4,4′,4′′-(4-Propyl-[1 H ]-pyrazole-1,3,5-triyl) tris phenol (Tocris, St. Louis, MO,USA,; ESR1 antagonist, MPP: 1,3-Bis(4-hydroxyphenyl)-4-methyl-5-[4-(piperidinylethoxy)phenol]-1H-pyrazol dihydrochloride (Tocris) (1μmol/L); ESR2 agonist, DPN: 2,3-bis(4-Hydroxyphenyl)-propionitrile) (Tocris) (100 nM); and 100 nM was used of the ESR2 antagonist: PHTPP, 4-[2-Phenyl-5,7-bis(trifluoromethyl) pyrazolo[1,5-a]pyrimidin-3-yl]phenol (Tocris).

Techniques: Control