Journal: OncoTargets and therapy

Article Title: Cholesterol biosynthesis inhibitor RO 48-8071 suppresses growth of hormone-dependent and castration-resistant prostate cancer cells

doi: 10.2147/OTT.S105725

Figure Lengend Snippet: Stimulation of ERβ activity potentiates the ability of RO to reduce viability of prostate cancer cells in vitro. Notes: PC-3 cells (4×10 3 /well) in 10% FBS RPMI-1640 medium were seeded in 96-well plates overnight and washed once with RPMI-1640 medium without FBS. Cells from RO alone and RO + DPN groups were pretreated with 15 μM RO for 2 hours and then treated with 15 μM RO alone or 15 μM RO +100 nM DPN for 22 hours. Cells of the DPN group were treated with 100 nM DPN alone for 22 hours without pretreatment (dose of DPN taken from previous studies , ). Cell viability was evaluated by SRB assay. * Significantly different from controls, ** significantly different from RO and DPN alone ( P <0.001, ANOVA). DPN alone was also significantly different from control at P =0.041 (ANOVA), representing inhibition of cell viability with low levels of ligand-bound endogenous ERβ (no RO treatment). Abbreviations: ANOVA, analysis of variance; DPN, diarylpropionitrile; ERβ, estrogen receptor β; FBS, fetal bovine serum; RO, 4′-(6-[allylmethylamino] hexyloxy)-4-bromo-2′-fluorobenzophenone fumarate; RPMI-1640 medium, Roswell Park Memorial Institute 1640 medium; SRB, sulforhodamine B.

Article Snippet: The ERβ agonist diarylpropionitrile (DPN) was obtained from Tocris Biosciences (Ellisville, MO, USA).

Techniques: Activity Assay, In Vitro, Sulforhodamine B Assay, Control, Inhibition

Journal: Science advances

Article Title: 27-Hydroxycholesterol acts on estrogen receptor α expressed by POMC neurons in the arcuate nucleus to modulate feeding behavior.

doi: 10.1126/sciadv.adi4746

Figure Lengend Snippet: Fig. 4. 27HC activates POMC neurons via ERα. (A to L) Immunofluorescent staining for c-Fos (red) in the arcuate nucleus of the hypothalamus (ARH) of female [(A) to (F)] and male [(G) to (L)] ERα-ZsGreen mice 90 min after a single bolus ICV infusion of vehicle or 27HC (0.1 nmol). White arrows point to dual-colored neurons with ERα (green) and c-Fos (red) expressed. (M and N) Summary of quantification per section of female and male mice (n = 3). (O) Schematic of POMC neurons in the ARH (POMCARH) treated with 27HC for electrophysiology recordings. (P) A micrographic image showing a recorded POMCARH (red) neuron of male mice POMC-Cre/Rosa26-LSL-tdTOMATO mice. (Q) Resting membrane potential (RM) of POMCARH neurons before and after 27HC treatment. (R) Summary of the responsive ratio of POMCARH neurons for all doses of 27HC. (S and T) The effects of 100 nM 27HC on RM before and after ERα antagonist BHPI (S) or ERβ antagonist PHTPP (T) incubation. The brain slices were first preincubated with presynaptic inhibitors 1 μM TTX (a voltage-gated sodium channel blocker) + 30 μM CNQX (an AMPA receptor antagonist) + 30 μM DAP-5 (an NMDA receptor antagonist) + 50 μM bicuculline (a GABA receptor antagonist). Results are shown as means ± SEM. [(M) and (N)] *P < 0.05 indicates statistical significance in unpaired t tests. [(Q), (S), and (T)] **P < 0.01 and ****P < 0.0001 indicate statistical significance in paired t tests.

Article Snippet: To test whether ERα or ERβ is necessary for the stimulatory effects of 27HC on POMCARH neurons, brain slices were treated with 100 nM 27HC in the presence of TTX, a cocktail of synaptic inhibitors, and either BHPI (50 μM), an ERα antagonist (catalog no. 5.38005, MilliporeSigma), or PHTPP (10 μM), an ERβ antagonist (catalog no. 2662, Tocris).

Techniques: Staining, Membrane, Incubation

Journal: Acta physiologica (Oxford, England)

Article Title: Roles of oestradiol receptor alpha and beta against hypertension and brain mitochondrial dysfunction under intermittent hypoxia in female rats.

doi: 10.1111/apha.13255

Figure Lengend Snippet: Body weight before and after exposure to CIH, and mean, systolic, and diastolic arterial pressured after exposure to CIH in ovariectomized female rats treated with vehicle (Veh), the ERα (PPT), or the ERβ (DPN) agonist and exposed to room air (AIR) or chronic intermittent hypoxia (CIH). All data are mean ± SD.

Article Snippet: During the surgery, the animals were implanted subcutaneously in the upper mid-dorsal region with an osmotic pump (Alzet®; model 2ML4 – flow of 60μl/day during 28 days) for continuous delivery of either vehicle (2-hydroxypropyl-β-cyclodextrin, Cayman Chemicals, Ann Arbor, MI, USA), the ERα agonist (propylpyraoletriol-PPT - 30μg/kg/day) or the ERβ agonist (diarylpropionitril-DPN, 100μg/kg/day - both from Tocris, Bio-Techne Canada, Oakville, ON).

Techniques:

Journal: Acta physiologica (Oxford, England)

Article Title: Roles of oestradiol receptor alpha and beta against hypertension and brain mitochondrial dysfunction under intermittent hypoxia in female rats.

doi: 10.1111/apha.13255

Figure Lengend Snippet: Typical recordings of mitochondrial respiration measured in permeabilized brain cortex samples from ovariectomized female rats treated with vehicle, and exposed to either room air (Veh AIR) or CIH (Veh CIH), and one ovariectomized female rat treated with the ERα agonist and exposed to CIH (PPT CIH). X-axis reflects the time from the start of the experiment, Y-axis depicts the O2 concentration in the recording chamber. O2 consumption corresponds to the slope of each step of the experiment. The different steps are indicated by the arrows showing the sequential addition of the sample, the substrates (respiratory state 2), ADP (state 3), oligomycin (state 4) and CCCP. Note the effect of CIH exposure on O2 consumption and the protective effect of the ERα agonist (PPT). The ERβ agonist (DPN – not shown) had a similar protective effect.

Article Snippet: During the surgery, the animals were implanted subcutaneously in the upper mid-dorsal region with an osmotic pump (Alzet®; model 2ML4 – flow of 60μl/day during 28 days) for continuous delivery of either vehicle (2-hydroxypropyl-β-cyclodextrin, Cayman Chemicals, Ann Arbor, MI, USA), the ERα agonist (propylpyraoletriol-PPT - 30μg/kg/day) or the ERβ agonist (diarylpropionitril-DPN, 100μg/kg/day - both from Tocris, Bio-Techne Canada, Oakville, ON).

Techniques: Concentration Assay

Journal: Acta physiologica (Oxford, England)

Article Title: Roles of oestradiol receptor alpha and beta against hypertension and brain mitochondrial dysfunction under intermittent hypoxia in female rats.

doi: 10.1111/apha.13255

Figure Lengend Snippet: Mitochondrial oxygen consumption during respiratory states 2, 3 and 4 measured on permeabilized brain cortex samples in ovariectomized female rats treated with vehicle (Veh), the ERα (PPT), or the ERβ (DPN) agonist and exposed to room air (AIR) or chronic intermittent hypoxia (CIH). A: NADH-linked mitochondrial respiration (complex I – activated by pyruvate and malate - PM). B: FADH2-linked mitochondrial respiration (complex II – activated by succinate and complex I blocked by rotenone - SR). D: NADH+FADH2-linked mitochondrial respiration (complexes I + II – activated by pyruvate + malate + succinate - PMS). All data are box and whiskers (median, 25th and 75th percentiles, min and max values). †, ††, ††† : p<0.01, p<0.001, and p 0.0001 vs Veh AIR.

Article Snippet: During the surgery, the animals were implanted subcutaneously in the upper mid-dorsal region with an osmotic pump (Alzet®; model 2ML4 – flow of 60μl/day during 28 days) for continuous delivery of either vehicle (2-hydroxypropyl-β-cyclodextrin, Cayman Chemicals, Ann Arbor, MI, USA), the ERα agonist (propylpyraoletriol-PPT - 30μg/kg/day) or the ERβ agonist (diarylpropionitril-DPN, 100μg/kg/day - both from Tocris, Bio-Techne Canada, Oakville, ON).

Techniques:

Journal: Acta physiologica (Oxford, England)

Article Title: Roles of oestradiol receptor alpha and beta against hypertension and brain mitochondrial dysfunction under intermittent hypoxia in female rats.

doi: 10.1111/apha.13255

Figure Lengend Snippet: Activity of citrate synthase (A), cytochrome-c oxidase (complex IV - B), and complex I (C) measured on brain cortex samples in ovariectomized female rats treated with vehicle (Veh), the ERα (PPT), or the ERβ (DPN) agonist and exposed to room air (AIR) or chronic intermittent hypoxia (CIH). All data are box and whiskers (median, 25th and 75th percentiles, min and max values) † : p<0.01, vs Veh AIR.

Article Snippet: During the surgery, the animals were implanted subcutaneously in the upper mid-dorsal region with an osmotic pump (Alzet®; model 2ML4 – flow of 60μl/day during 28 days) for continuous delivery of either vehicle (2-hydroxypropyl-β-cyclodextrin, Cayman Chemicals, Ann Arbor, MI, USA), the ERα agonist (propylpyraoletriol-PPT - 30μg/kg/day) or the ERβ agonist (diarylpropionitril-DPN, 100μg/kg/day - both from Tocris, Bio-Techne Canada, Oakville, ON).

Techniques: Activity Assay

Journal: Acta physiologica (Oxford, England)

Article Title: Roles of oestradiol receptor alpha and beta against hypertension and brain mitochondrial dysfunction under intermittent hypoxia in female rats.

doi: 10.1111/apha.13255

Figure Lengend Snippet: Activity of pyruvate dehydrogenase (A), lactate dehydrogenase (B), and activity ratio of lactate dehydrogenase to pyruvate dehydrogenase (LDH/PDH - C) on brain cortex samples in ovariectomized female rats treated with vehicle (Veh), the ERα (PPT), or the ERβ (DPN) agonist and exposed to room air (AIR) or chronic intermittent hypoxia (CIH). All data are box and whiskers (median, 25th and 75th percentiles, min and max values) *, † , ††, ††† : p<0.05, p<0.01, p<0.001, and p<0.0001 vs Veh AIR.

Article Snippet: During the surgery, the animals were implanted subcutaneously in the upper mid-dorsal region with an osmotic pump (Alzet®; model 2ML4 – flow of 60μl/day during 28 days) for continuous delivery of either vehicle (2-hydroxypropyl-β-cyclodextrin, Cayman Chemicals, Ann Arbor, MI, USA), the ERα agonist (propylpyraoletriol-PPT - 30μg/kg/day) or the ERβ agonist (diarylpropionitril-DPN, 100μg/kg/day - both from Tocris, Bio-Techne Canada, Oakville, ON).

Techniques: Activity Assay

Journal: Acta physiologica (Oxford, England)

Article Title: Roles of oestradiol receptor alpha and beta against hypertension and brain mitochondrial dysfunction under intermittent hypoxia in female rats.

doi: 10.1111/apha.13255

Figure Lengend Snippet: Concentration of proteins in brain samples lysates in ovariectomized female rats treated with vehicle (Veh), the ERα (PPT), or the ERβ (DPN) agonist and exposed to room air (AIR) or chronic intermittent hypoxia (CIH). All data are mean ± SD

Article Snippet: During the surgery, the animals were implanted subcutaneously in the upper mid-dorsal region with an osmotic pump (Alzet®; model 2ML4 – flow of 60μl/day during 28 days) for continuous delivery of either vehicle (2-hydroxypropyl-β-cyclodextrin, Cayman Chemicals, Ann Arbor, MI, USA), the ERα agonist (propylpyraoletriol-PPT - 30μg/kg/day) or the ERβ agonist (diarylpropionitril-DPN, 100μg/kg/day - both from Tocris, Bio-Techne Canada, Oakville, ON).

Techniques: Concentration Assay

Journal: Journal of Biomedical Science

Article Title: Activation of estrogen receptor β-dependent nitric oxide signaling mediates the hypotensive effects of estrogen in the rostral ventrolateral medulla of anesthetized rats

doi: 10.1186/1423-0127-16-60

Figure Lengend Snippet: Effects of ER antagonist on the E2β-induced cardiovascular depressive responses . Time-course of the changes in MSAP and total power density of vasomotor components (0–0.8 Hz) of SAP spectrum in anaesthetized rats that received microinjection bilaterally into the RVLM (at time 0) of aCSF, or E2β (1 pmol) given together with ICI 182780 (ICI, 0.25 or 0.5 pmol) or 5% DMSO. Values are presented as mean ± S.E.M., n = 6–8 animals per experimental group. *P < 0.05 versus corresponding aCSF group, and # P < 0.05 versus corresponding E2β+DMSO group in the Scheffé multiple range test.

Article Snippet: The test agents were used in this study included 17β-estradiol-3-sulphate sodium (E2β; Sigma-Aldrich, St. Louis, MO, USA); a selective ERα agonist, 1,3,5- tris (4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT; Tocris Cookson Inc., Bristol, UK); a selective ERβ agonist, 2,3- bis (4-hydroxyphenyl) propionitrile (DPN; Tocris Cookson); 17α-estradiol (E2α; Sigma-Aldrich); a nonspecific ER antagonist, ICI 182780 (Tocris Cookson); a selective ERα antagonist, methyl-piperidino-pyrazole (MPP; Tocris Cookson); a selective ERβ antagonist, R,R-tetrahydrochrysene (R,R-THC; Tocris Cookson); a nonselective NO synthase (NOS) inhibitor, N G -nitro-L-arginine methyl ester (L-NAME; Sigma-Aldrich); a selective inducible NOS (iNOS) inhibitor, S-methylisothiourea (SMT; Tocris Cookson); a selective neuronal NOS (nNOS) inhibitor, 7-nitroindazole (7-NI; Tocris Cookson); a selective endothelial NOS (eNOS) inhibitor, N5-(1-Iminoethyl)-L-ornithine (L-NIO; Tocris Cookson); or a transcription inhibitor, actinomycin D (AMD; Tocris Cookson).

Techniques: Microinjection

Journal: Journal of Biomedical Science

Article Title: Activation of estrogen receptor β-dependent nitric oxide signaling mediates the hypotensive effects of estrogen in the rostral ventrolateral medulla of anesthetized rats

doi: 10.1186/1423-0127-16-60

Figure Lengend Snippet: Cardiovascular effects after microinjection bilaterally into the RVLM of ERα or ERβ agonist . Time-course of the changes in MSAP, HR and total power density of vasomotor components (0–0.8 Hz) of SAP spectrum in anaesthetized rats that received microinjection bilaterally into the RVLM (at time 0) of aCSF, DPN (1, 2 or 5 pmol), or PPT (5 pmol). Values are presented as mean ± S.E.M., n = 6–8 animals per experimental group. * P < 0.05 versus corresponding aCSF group in the Scheffé multiple range test.

Article Snippet: The test agents were used in this study included 17β-estradiol-3-sulphate sodium (E2β; Sigma-Aldrich, St. Louis, MO, USA); a selective ERα agonist, 1,3,5- tris (4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT; Tocris Cookson Inc., Bristol, UK); a selective ERβ agonist, 2,3- bis (4-hydroxyphenyl) propionitrile (DPN; Tocris Cookson); 17α-estradiol (E2α; Sigma-Aldrich); a nonspecific ER antagonist, ICI 182780 (Tocris Cookson); a selective ERα antagonist, methyl-piperidino-pyrazole (MPP; Tocris Cookson); a selective ERβ antagonist, R,R-tetrahydrochrysene (R,R-THC; Tocris Cookson); a nonselective NO synthase (NOS) inhibitor, N G -nitro-L-arginine methyl ester (L-NAME; Sigma-Aldrich); a selective inducible NOS (iNOS) inhibitor, S-methylisothiourea (SMT; Tocris Cookson); a selective neuronal NOS (nNOS) inhibitor, 7-nitroindazole (7-NI; Tocris Cookson); a selective endothelial NOS (eNOS) inhibitor, N5-(1-Iminoethyl)-L-ornithine (L-NIO; Tocris Cookson); or a transcription inhibitor, actinomycin D (AMD; Tocris Cookson).

Techniques: Microinjection

Journal: Journal of Biomedical Science

Article Title: Activation of estrogen receptor β-dependent nitric oxide signaling mediates the hypotensive effects of estrogen in the rostral ventrolateral medulla of anesthetized rats

doi: 10.1186/1423-0127-16-60

Figure Lengend Snippet: Effects of NOS inhibitor on the ERβ agonist-induced cardiovascular depressive effects . Time-course of the changes in MSAP and total power density of vasomotor components (0–0.8 Hz) of SAP spectrum in anaesthetized rats that received microinjection bilaterally into the RVLM (at time 0) of aCSF, 3% methanol (MeOH), or DPN (2 pmol) given together with L-NAME (5 nmol), SMT (25 pmol), 7-NI (0.5 pmol), L-NIO (2.5 pmol) or 3% MeOH. Values are mean ± S.E.M., n = 6–7 animals per experimental group. * P < 0.05 versus corresponding MeOH group, and # P < 0.05 versus corresponding DPN+MeOH group in the Scheffé multiple range test. Data on aCSF are not shown because they in essence duplicated those by 3% MeOH.

Article Snippet: The test agents were used in this study included 17β-estradiol-3-sulphate sodium (E2β; Sigma-Aldrich, St. Louis, MO, USA); a selective ERα agonist, 1,3,5- tris (4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT; Tocris Cookson Inc., Bristol, UK); a selective ERβ agonist, 2,3- bis (4-hydroxyphenyl) propionitrile (DPN; Tocris Cookson); 17α-estradiol (E2α; Sigma-Aldrich); a nonspecific ER antagonist, ICI 182780 (Tocris Cookson); a selective ERα antagonist, methyl-piperidino-pyrazole (MPP; Tocris Cookson); a selective ERβ antagonist, R,R-tetrahydrochrysene (R,R-THC; Tocris Cookson); a nonselective NO synthase (NOS) inhibitor, N G -nitro-L-arginine methyl ester (L-NAME; Sigma-Aldrich); a selective inducible NOS (iNOS) inhibitor, S-methylisothiourea (SMT; Tocris Cookson); a selective neuronal NOS (nNOS) inhibitor, 7-nitroindazole (7-NI; Tocris Cookson); a selective endothelial NOS (eNOS) inhibitor, N5-(1-Iminoethyl)-L-ornithine (L-NIO; Tocris Cookson); or a transcription inhibitor, actinomycin D (AMD; Tocris Cookson).

Techniques: Microinjection

Journal: BMJ Open Diabetes Research & Care

Article Title: Insulin receptor cleavage induced by estrogen impairs insulin signaling

doi: 10.1136/bmjdrc-2021-002467

Figure Lengend Snippet: Increased calpain 2 release in exosomes via GPER. (A) IR cleavage by chemical compounds specific for estrogen receptor subtypes. HepG2 cells were incubated with the indicated concentrations of chemical compounds for 48 hours, followed by an additional 24 hours before preparation of media (n=9–18). (B) G1-induced IR cleavage by calpain 2. Following knockdown of calpain 2, cells were treated with 2.5 µmol/L G1 for 48 hours. Subsequently, cells were incubated for an additional 24 hours before preparation of media (n=14–17). (C) Estradiol-induced IR cleavage via GPER. Following knockdown of GPER, cells were treated with 40 µmol/L estradiol for 48 hours. Subsequently, cells were incubated for an additional 24 hours before preparation of media (n=9). (D) G1-induced IR cleavage in a dose-dependent manner. HepG2 cells were incubated with the indicated concentrations of G1 for 48 hours, followed by an additional 24 hours before preparation of media (n=9). (E) Assessment of time dependence of G1-induced IR cleavage. HepG2 cells were incubated with 2.5 µmol/L G1 for 0, 24, or 48 hours. The graph shows the sIR levels for an additional 24 hours (n=9). (F) Assessment of reversibility of G1-induced IR cleavage. HepG2 cells were incubated with 2.5 µmol/L G1 for 48 hours, after which the medium was replaced with control for 0, 24, or 48 hours. The graph shows the sIR levels for an additional 24 hours (n=9). (G) Pharmacological inhibition of IR cleavage using metformin. Following incubation with 2.5 µmol/L G1 and the indicated concentrations of metformin for 48 hours, cells were incubated for an additional 24 hours before preparation of media (n=14–32). (H) Enhanced calpain 2 release in exosomes and its inhibition by metformin. Following incubation with 2 µmol/L G1 and 2 mmol/L metformin for 48 hours, cells were incubated in serum-free DMEM for an additional 24 hours, and then exosomes from media and cell lysates were prepared. (I) Enhanced calpain 2 protein expression by GPER signaling. Following incubation with 2 µmol/L G1 for 48 hours, cells were incubated in serum-free DMEM for an additional 24 hours and then cell lysates were prepared. The graph shows the statistical analysis of the experiments (n=5). (J) Enhanced expression of calpain 2 mRNA by GPER signaling. HepG2 cells were incubated with 2 µmol/L G1 for 72 hours before preparation of mRNA. The graph shows the statistical analysis of the experiments (n=18). (K) Estradiol-induced calpain 2 expression via GPER. Following knockdown of GPER, cells were incubated with 40 µmol/L estradiol for 72 hours before preparation of the cell lysates. *P<0.05, **P<0.01, ***P<0.001, n.s., not significant. DMEM, Dulbecco’s modified eagle medium; DPN, diarylpropionitrile; GPER, G protein-coupled estrogen receptor; IR, insulin receptor; mRNA, messenger RNA; PPT, propylpyrazole triol; sIR, soluble insulin receptor; siRNA, small interfering RNA.

Article Snippet: Propylpyrazole triol (PPT) (estrogen receptor (ER) α selective agonist) and diarylpropionitrile (DPN) (ERβ selective agonist) were purchased from Tocris Bioscience (Bristol, UK).

Techniques: Incubation, Knockdown, Control, Inhibition, Expressing, Modification, Small Interfering RNA