Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

Article Title: 2-Phenylacetamide Isolated from the Seeds of Lepidium apetalum and Its Estrogen-Like Effects In Vitro and In Vivo

doi: 10.3390/molecules23092293

Figure Lengend Snippet: Western blot analysis of the expression of ER α , ER β and GPR30 in the uterus of immature female Swiss mice and MCF-7 cells ( n = 3). Figure ( A ), ( B ), ( C ) and ( D ) show the expression of ER α , ER β and GPR30 in the uterus. Figure ( E ), ( F ), ( G ) and ( H ) show the expression of ER α , ER β and GPR30 in MCF-7 cells. * p < 0.05; ** p < 0.01 compared to controls.

Article Snippet: The ER-unspecific antagonist ICI182780 (Tocris, Bristol, UK; 1 μM), the specific ER α antagonist methylpiperidino-pyrazole (MPP; Tocris, Bristol, UK; 1 μM), the specific ER β antagonist Delta (9)-tetrahydrocannabinol (THC; Tocris, Bristol, UK; 1 μM) and the seven-transmembrane domain protein G protein-coupled receptor (G15; Tocris, Bristol, UK; 1 μM) were added 30 min before treatment of 17 β -E2, LA or PA. Other experimental steps are as described above in .

Techniques: Western Blot, Expressing

Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

Article Title: 2-Phenylacetamide Isolated from the Seeds of Lepidium apetalum and Its Estrogen-Like Effects In Vitro and In Vivo

doi: 10.3390/molecules23092293

Figure Lengend Snippet: Western blot analysis of the expression of estrogen receptors in the uterus of immature female Swiss mice and MCF-7 cells ( n = 3). Figure ( A ), ( B ), ( C ) and ( D ) show the expression of ER α , ER β and GPR30 in the uterus. Figure ( E ), ( F ), ( G ) and ( H ) show the expression of ER α , ER β and GPR30 in MCF-7 cells. * p < 0.05; ** p < 0.01 compared to controls.

Article Snippet: The ER-unspecific antagonist ICI182780 (Tocris, Bristol, UK; 1 μM), the specific ER α antagonist methylpiperidino-pyrazole (MPP; Tocris, Bristol, UK; 1 μM), the specific ER β antagonist Delta (9)-tetrahydrocannabinol (THC; Tocris, Bristol, UK; 1 μM) and the seven-transmembrane domain protein G protein-coupled receptor (G15; Tocris, Bristol, UK; 1 μM) were added 30 min before treatment of 17 β -E2, LA or PA. Other experimental steps are as described above in .

Techniques: Western Blot, Expressing

Journal: Cell Death & Disease

Article Title: Neuroglobin, a pro-survival player in estrogen receptor α -positive cancer cells

doi: 10.1038/cddis.2014.418

Figure Lengend Snippet: Impact of estrogen receptors on NGB protein expression in HeLa, HepG2, and MCF-7 cells. Western blot of ER α and ER β levels in non-stimulated cells compared with 5 ng of recombinant proteins in ( a ) HepG2 cells and ( b ) MCF-7 cells. ( c ) Western blot of NGB levels in HepG2 cells stimulated for 24 h with vehicle, E2 (10 nM), ER β agonist DPN (10 nM), ER α agonist PPT (10 nM) or the ER inhibitor ICI (1 μ M) in the absence and presence of E2 (10 nM). ( d ) Western blot of NGB levels in HepG2 cells stimulated for 24 h with vehicle, E2 (10 nM) or ER β inhibitor THC (1 μ M) in the absence and presence of E2 (10 nM). ( e ) Western blot of NGB levels in MCF-7 cells treated for 24 h with vehicle, E2 (10 nM), ER β inhibitor THC (1 μ M) or ER inhibitor ICI (1 μ M) in the absence and presence of E2 (10 nM). ( f ) Western blot of NGB levels in MCF-7 cells stimulated for 24 h with vehicle, E2 (10 nM), ER β agonist DPN (10 nM) or ER α agonist PPT (10 nM). The amount of proteins was normalized by comparison with tubulin or vinculin levels. ( a , b ) The data are typical western blots of three independent experiments. ( c – f ) Top panels are typical western blots, bottom panels represent the results of the densitometric analysis. Data are means±S.D. of three different experiments. P <0.001 was determined with ANOVA followed by Tukey–Kramer post-test versus vehicle (*) and versus E2-treated sample (°)

Article Snippet: The E2 antagonist ICI, ER α -selective agonist PPT, ER β -selective agonist DPN, and ER β -selective antagonist THC were obtained from Tocris (Ballwin, MO, USA).

Techniques: Expressing, Western Blot, Recombinant, Comparison

Journal: Journal of Dental Research

Article Title: 17β-estradiol Induces MMP-9 and MMP-13 in TMJ Fibrochondrocytes via Estrogen Receptor α

doi: 10.1177/0022034518767108

Figure Lengend Snippet: 17β-estradiol (E2) upregulates matrix metalloproteinase (MMP) expression via estrogen receptor α (ERα) but not ERβ in mouse temporomandibular joint (TMJ) fibrochondrocytes. Untransfected cells (controls) or cells transiently transfected with control siRNA (C-siRNA) or siRNA to ERs were cultured in the absence or presence of E2. Western blot shows suppression of ERα (A) or ERβ (B) in TMJ fibrochondrocytes transfected with ERα- or ERβ-specific siRNAs, respectively. (C, D) Representative images and pooled data in the graph show that E2’s induction of MMP-9 and MMP-13 was abrogated in cells in which ERα expression was suppressed. (E, F) Representative images and pooled data in the graph demonstrate that inhibition of ERβ expression did not change E2-induced MMP-9 and MMP-13 levels. (G, H) The cells treated with pan-ER-antagonist (ICI) or ERα inhibitor (MPP) or ERβ inhibitor (PHTPP) and then stimulated with 0.5 ng/mL of E2 show that the induction of MMP-9 is inhibited in the presence of ICI and MPP but not by PHTPP. Actin was used as a loading control. **P < 0.01 vs. corresponding vehicle-treated control. ##P < 0.01 vs. control within E2 concentration treatment group. ⌘⌘P < 0.01 vs. corresponding E2 treatment group (0.1 ng/mL). Values are presented as mean ± SD fold change.

Article Snippet: To determine the effect of ER signaling on MMP expression, the fibrochondrocytes were transfected with 250pM extracellular signal-regulated kinase 1/2 (ERK), E-26 transcription factor (ELK-1), or p65 nuclear factor kappa B (NF-κB; a major unit of NF-κB) or control siRNAs (sc-29859, sc-3529, sc-29411, and sc-37007; Santa Cruz Biotech) or treated with the following signaling inhibitors: 10μM U0126 (Santa Cruz Biotech), 1μM MPP for ERα, 1μM PHTPP for ERβ and 1μM ICI for ERα and ERβ (Tocris Biosciences) for 30 min prior to incubation with E2.

Techniques: Expressing, Transfection, Cell Culture, Western Blot, Inhibition, Concentration Assay

Journal: Journal of Dental Research

Article Title: 17β-estradiol Induces MMP-9 and MMP-13 in TMJ Fibrochondrocytes via Estrogen Receptor α

doi: 10.1177/0022034518767108

Figure Lengend Snippet: Estrogen receptor α (ERα) overexpression enhances matrix metalloproteinase 9 (MMP-9) expression but does not modulate MMP-13 expression by 17β-estradiol (E2) in mouse temporomandibular joint (TMJ) fibrochondrocytes, while ERβ overexpression does not affect E2-induced MMP-9 or MMP-13 expression. Untransfected cells (controls) or cells transiently transfected with pcDNA vector or ERα or ERβ cDNA constructs were cultured in the absence or presence of E2. Western blots of TMJ cell lysates showed overexpression of ERα (A) or ERβ (B) following transfection of cells with their respective pcDNA vectors. (C, D) Representative images and pooled graphical data show that overexpression of ERα increases the expression of MMP-9 induced by E2, with no effect on E2’s modulation of MMP-13 levels. (E, F) Representative images and pooled graphical data demonstrate that overexpression of ERβ does not modulate MMP-9 or MMP-13 expression by E2. Actin was used as an internal control. **P < 0.01 vs. corresponding vehicle-treated control. ##P < 0.01 vs. corresponding within E2 treatment controls. Values are presented as mean ± SD fold change.

Article Snippet: To determine the effect of ER signaling on MMP expression, the fibrochondrocytes were transfected with 250pM extracellular signal-regulated kinase 1/2 (ERK), E-26 transcription factor (ELK-1), or p65 nuclear factor kappa B (NF-κB; a major unit of NF-κB) or control siRNAs (sc-29859, sc-3529, sc-29411, and sc-37007; Santa Cruz Biotech) or treated with the following signaling inhibitors: 10μM U0126 (Santa Cruz Biotech), 1μM MPP for ERα, 1μM PHTPP for ERβ and 1μM ICI for ERα and ERβ (Tocris Biosciences) for 30 min prior to incubation with E2.

Techniques: Over Expression, Expressing, Transfection, Plasmid Preparation, Construct, Cell Culture, Western Blot

Journal: Journal of Dental Research

Article Title: 17β-estradiol Induces MMP-9 and MMP-13 in TMJ Fibrochondrocytes via Estrogen Receptor α

doi: 10.1177/0022034518767108

Figure Lengend Snippet: Estrogen receptor α (ERα) and ERβ differentially regulate 17β-estradiol (E2)–induced phosphorylation of the ERK, which mediates matrix metalloproteinase 9 (MMP-9) induction in temporomandibular joint (TMJ) fibrochondrocytes. (A, B) Knockdown of ERα diminishes, and knockdown of ERβ enhances, E2-stimulated phosphorylation levels of ERK. (C) Immunofluorescence for pERK showing that E2 stimulation resulted in increased pERK staining (red), which was diminished in cells transfected with ERα siRNA and increased in cells transfected with ERβ siRNA relative to untransfected or C-siRNA-transfected controls. (D) E2-stimulated phosphorylation of ERK was inhibited by ICI, MPP, and pERK activation inhibitor U0126 but augmented by PHTPP. (E, F) Overexpression of ERα increased whereas overexpression of ERβ reduced the phosphorylation of ERK following stimulation with E2, as determined by Western blots and immunofluorescence. White bar in the lower right image in panel F represents 100 µm. (G, H) Western blot and pooled graphical data demonstrate that suppression of ERK by its siRNA abolished E2-induced upregulation of MMP-9. Actin was used as an internal control. *P < 0.05 and **P < 0.01 vs. corresponding vehicle-treated controls. ##P < 0.01 vs. corresponding within E2 treatment control and C-siRNA controls. Values are presented as mean ± SD fold change.

Article Snippet: To determine the effect of ER signaling on MMP expression, the fibrochondrocytes were transfected with 250pM extracellular signal-regulated kinase 1/2 (ERK), E-26 transcription factor (ELK-1), or p65 nuclear factor kappa B (NF-κB; a major unit of NF-κB) or control siRNAs (sc-29859, sc-3529, sc-29411, and sc-37007; Santa Cruz Biotech) or treated with the following signaling inhibitors: 10μM U0126 (Santa Cruz Biotech), 1μM MPP for ERα, 1μM PHTPP for ERβ and 1μM ICI for ERα and ERβ (Tocris Biosciences) for 30 min prior to incubation with E2.

Techniques: Immunofluorescence, Staining, Transfection, Activation Assay, Over Expression, Western Blot