Journal: International journal of molecular sciences

Article Title: Isoflavones Mediate Dendritogenesis Mainly through Estrogen Receptor α.

doi: 10.3390/ijms24109011

Figure Lengend Snippet: Figure 2. ER and GPER1 are essential for isoflavone-augmented dendrite arborization in Purkinje cells. Cerebellar cells were cultured for 17 days, followed by an immunocytochemistry analysis of Purkinje cells with Calbindin D-28K (red) and DAPI (blue). (A) Representative photomicrographs showing the co-exposure effects of genistein with ICI 182,780 or G15 on the morphology of Purkinje cells. (B) Changes in the dendritic areas in Purkinje cells following S-equol, G15, and/or ICI 182,780 treatment (n = 15). (C) Representative photomicrographs showing the effects of genistein on the morphology of Purkinje cells after ERα, ERβ, or GPER1 knockdown. Quantitative analysis of the effects of isoflavones after exposure to ERα siRNA (n = 15) (D), ERβ siRNA (n = 15) (E), siRNA against ERα and ERβ (n = 15) (F), and GPER1 siRNA (n = 15) (G) on the dendritic areas in Purkinje cells following isoflavone treatment. Bars indicate 50 µm. Data are shown as the mean ± standard error of the mean and represent at least three independent experiments. #### p < 0.0001 indicates statistical significance according to two-way or one-way analysis of variance, followed by the post hoc Tukey test compared with E2 (10 nM). **** p < 0.0001 and *** p < 0.001 indicate statistical significance according to two-way or one-way analysis of variance, followed by the post hoc Tukey test.

Article Snippet: Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20, followed by overnight incubation with the appropriate diluted primary antibodies for ERα (1:1000; MerckMilipore, CA, USA; catalog number: 06935), ERβ (1:1000; GeneTex, Inc, CA, USA; catalog number: GTX70174), GPER1 (1:1000; Alomone Labs, JBP, Israel; catalog number: AER050), and GAPDH (1:1000; Proteintech, IL, USA; catalog number: 600041Ig).

Techniques: Cell Culture, Immunocytochemistry, Knockdown

Journal: International journal of molecular sciences

Article Title: Isoflavones Mediate Dendritogenesis Mainly through Estrogen Receptor α.

doi: 10.3390/ijms24109011

Figure Lengend Snippet: Figure 3. ERα affects isoflavone-induced neurite outgrowth in neuronal-derived Neuro-2A cells. Cells were induced to differentiate through serum starvation for 1–3 days, followed by immunofluo- rescence analysis with β-tubulin III (blue), doublecortin (red), and F-actin (green). (A) Representa- tive photomicrographs showing the effects of isoflavones on the differentiation of Neuro-2A cells. (B) Changes in neurite lengths after exposure to isoflavones (n = 30). Quantitative analysis of the effect of isoflavones after exposure to ICI 182,780 (C), G15 (D) ERa siRNA (E), ERb siRNA (F), ERα and ERβ siRNA (G), and GPER1 siRNA (H) on neurite length in Neuro-2A cells (n = 30). Bars indicate 50 µm. Data are shown as the mean ± standard error of the mean and represent at least three independent experiments. #### p < 0.0001 indicates statistical significance according to two-way or one-way analysis of variance, followed by the post hoc Tukey test compared to E2 (10 nM). **** p < 0.0001, *** p < 0.001, ** p < 0.01 and * p < 0.05 indicate statistical significance according to two-way or one-way analysis of variance, followed by the post hoc Tukey test.

Article Snippet: Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20, followed by overnight incubation with the appropriate diluted primary antibodies for ERα (1:1000; MerckMilipore, CA, USA; catalog number: 06935), ERβ (1:1000; GeneTex, Inc, CA, USA; catalog number: GTX70174), GPER1 (1:1000; Alomone Labs, JBP, Israel; catalog number: AER050), and GAPDH (1:1000; Proteintech, IL, USA; catalog number: 600041Ig).

Techniques: Derivative Assay

Journal: International journal of molecular sciences

Article Title: Isoflavones Mediate Dendritogenesis Mainly through Estrogen Receptor α.

doi: 10.3390/ijms24109011

Figure Lengend Snippet: Figure 4. Isoflavone-induced morphological changes in neuronal-derived Neuro-2A cells through the modulation of ERα. Cells were induced to differentiate through serum starvation for 1–3 days, followed by immunofluorescence analysis with β-tubulin III (blue), doublecortin (red), and F-actin (green). (A) Diagram of Sholl analysis. (B) Quantitative analysis of the effect of isoflavones on the number of neurite intersections at each concentric circle by Sholl analysis. Effects of genistein (C), daidzein (D), S-equol (E), or E2 (F) on the number of neurite intersections in Neuro-2A cells after the exposure of DsiRNA, siRNA against ERα, ERβ, or GPER1 (n = 30). (G) Representative images of blots for ERα, ERβ, GPER1, or GAPDH levels in response to isoflavone exposure in Neuro-2A cells. Quantitative analysis of the effect of isoflavones on ERα (H), ERβ (I), and GPER1 protein expression levels (J) (n = 6). Bars indicate 50 µm. Data are shown as the mean ± standard error of the mean and represent at least three independent experiments. **** p < 0.0001 and *** p < 0.001 indicate statistical significance according to two-way or one-way analysis of variance, followed by the post hoc Tukey test.

Article Snippet: Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20, followed by overnight incubation with the appropriate diluted primary antibodies for ERα (1:1000; MerckMilipore, CA, USA; catalog number: 06935), ERβ (1:1000; GeneTex, Inc, CA, USA; catalog number: GTX70174), GPER1 (1:1000; Alomone Labs, JBP, Israel; catalog number: AER050), and GAPDH (1:1000; Proteintech, IL, USA; catalog number: 600041Ig).

Techniques: Derivative Assay, Immunofluorescence, Expressing

Journal: Open Journal of Molecular and Integrative Physiology

Article Title: Effects of selective estrogen receptor agonists on estrogen receptor expression in the uterus of ovariectomized rats

doi: 10.4236/ojmip.2012.22006

Figure Lengend Snippet: Figure 2. A representative western blot of ERα, ERβ and GPER in uterus from E2, PPT and DPN treated rats and the OvxC control group. β-Actin was used as loading control.

Article Snippet: An anti-human GPER polyclonal antibody (HPA027052) raised in rabbit was purchased from Atlas antibodies, Sweden.

Techniques: Western Blot, Control

Journal: Open Journal of Molecular and Integrative Physiology

Article Title: Effects of selective estrogen receptor agonists on estrogen receptor expression in the uterus of ovariectomized rats

doi: 10.4236/ojmip.2012.22006

Figure Lengend Snippet: Figure 1. The mRNA levels of ERα, ERβ and GPER in the rat uterus. OvxC = ovariectomized controls; E2, PPT and DPN treated rats; n = 8 in all groups but DPN where it is 6. Bars with an asterisk show sig- nificant difference to the control group (OvxC) (p < 0.05).

Article Snippet: An anti-human GPER polyclonal antibody (HPA027052) raised in rabbit was purchased from Atlas antibodies, Sweden.

Techniques: Control

Journal: Open Journal of Molecular and Integrative Physiology

Article Title: Effects of selective estrogen receptor agonists on estrogen receptor expression in the uterus of ovariectomized rats

doi: 10.4236/ojmip.2012.22006

Figure Lengend Snippet: Figure 3. Representative images from immunohistochemistry staining of ERα (A)-(D), ERβ (E)-(H), GPER (I)-(L), Ki-67 (M)-(P) and control slides (Q)-(U). LE = luminal epithelium, GE = glandular epithe- lium, Str = stroma, Myo = myometrium and GC = granulosa cells. The bottom panel shows a negative control for ERα IHC in rat uterus (Q), a positive control for ERβ in rat ovary (R), a negative control for ERβ in rat ovary (S), and negative controls for GPER (T) and Ki-67 (U) in rat uterus. The images from the different treatment groups are placed in columns for image (A)-(P); OvxC (Column 1), E2 (Column 2), PPT (Column 3) and DPN (Column 4). Magnification ×400, the bar represents 50 µm.

Article Snippet: An anti-human GPER polyclonal antibody (HPA027052) raised in rabbit was purchased from Atlas antibodies, Sweden.

Techniques: Immunohistochemistry, Staining, Control, Negative Control, Positive Control

Journal: Scientific Reports

Article Title: AOH induces oxidative stress and DNA damage in ovarian cancer cells via modulation of GPER1 and HIF1α/PI3K/CLDNs signaling pathway

doi: 10.1038/s41598-025-26042-9

Figure Lengend Snippet: AOH decreases the viability of OVCAR3 cells and modulates the expression of GPER1. ( a ) Viability of OVCAR3 cells: The viability of cells after treatment with AOH for 24 h (MTT assay, the results are expressed as % of control (non-treated cells)). ( b ) GPER1 gene expression: Relative expression of GPER1 after 24 h with AOH ( c ) GPER1 protein expression: Western Blot analysis of GPER1 expression after 24 h with AOH, the representative results. All the results are expressed as mean ± SE. One-way ANOVA with Bonferroni correction was used as the statistical analysis. P < 0.05 was considered statistically significant, * p < 0.05, ** p < 0.01, *** p < 0.001. AOH alternariol, GPER1 G protein-coupled estrogen receptor 1, Vin vinculin, Cnt control (non-treated cells).

Article Snippet: Akt (#9272), phospho-Akt (Ser473) (#4060), PARP1 (#46,011), SOD1 (#4266), SOD2 (#13,141), pH2AX (#9718), CLDN1 (#13,995), CLDN3 (#83,609), PI3K (#11,889), p44/42 (#4695), phospho-p44/42 (#4370), CLDN4 (STJ114778), GPER1 (STJ192629) (St John’s Laboratory Ltd, University Way, London) antibodies were used according to the manufacturer’s recommendations.

Techniques: Expressing, MTT Assay, Control, Gene Expression, Western Blot

Journal: Scientific Reports

Article Title: AOH induces oxidative stress and DNA damage in ovarian cancer cells via modulation of GPER1 and HIF1α/PI3K/CLDNs signaling pathway

doi: 10.1038/s41598-025-26042-9

Figure Lengend Snippet: AOH induces oxidative stress in OVCAR3 cells. ( a ) Oxidative stress: The analysis of the generation of ROS with flow cytometry. ( b ) Representative results of flow cytometry. ( c ) SOD2 protein expression: Expression of SOD2 was evaluated with Western Blot, the representative results. ( d ) SOD1, SOD2, PARP1 and XRCC1 gene expression: Relative expression of SOD1, SOD2 , PARP1 and XRCC1 . ( e ) DNA damage: evaluated with flow cytometry (ATM and H2A.X activation). ( f ) Representative results of flow cytometry. ( g ) p-H2A.X, PARP1 and cleaved-PARP1 protein expression: Expression of phospho-H2A.X, PARP1 and cleaved—PARP1 evaluated with Western Blot, the representative results. ( h ) Confocal microscopy: Bar graph showing the difference in normalized mean fluorescence intensity signal. ( i ) Immunofluorescent staining of phospho-H2A.X (green) and nucleus (orange, NucSpot 555 / 570). All the results are expressed as mean ± SE. One-way ANOVA with Bonferroni correction was used for statistical analysis. P < 0.05 was considered statistically significant, * p < 0.05, ** p < 0.01, *** p < 0.001. AOH alternariol, G15 selective GPER1 receptor antagonist, G1 selective GPER1 agonist, SOD1 Superoxide dismutase 1, SOD2 Superoxide dismutase 2, PARP1 Poly [ADP-ribose] polymerase 1, XRCC1 X-ray repair cross-complementing protein 1, GAPDH anti-glyceraldehyde 3-phosphate dehydrogenase, pH2AX phospho-H2A histone family member X. Cnt control (non-treated cells).

Article Snippet: Akt (#9272), phospho-Akt (Ser473) (#4060), PARP1 (#46,011), SOD1 (#4266), SOD2 (#13,141), pH2AX (#9718), CLDN1 (#13,995), CLDN3 (#83,609), PI3K (#11,889), p44/42 (#4695), phospho-p44/42 (#4370), CLDN4 (STJ114778), GPER1 (STJ192629) (St John’s Laboratory Ltd, University Way, London) antibodies were used according to the manufacturer’s recommendations.

Techniques: Flow Cytometry, Expressing, Western Blot, Gene Expression, Activation Assay, Confocal Microscopy, Fluorescence, Staining, Control

Journal: Scientific Reports

Article Title: AOH induces oxidative stress and DNA damage in ovarian cancer cells via modulation of GPER1 and HIF1α/PI3K/CLDNs signaling pathway

doi: 10.1038/s41598-025-26042-9

Figure Lengend Snippet: AOH modulates the expression of HIF1α , Akt and claudins . ( a ) HIF1α gene expression: Relative expression of HIF1α. ( b ) CLDN1, CLDN3 and CLDN3 protein expression: Western Blot analysis of CLDN1, CLDN3 and CLDN3, the representative results. ( c ) CLDN1, CLDN3 and CLDN3 gene expression: Relative expression of CLDN1. CLDN3 and CLDN4 . ( d ) pAkt, Akt and PI3K protein expression: Western Blot analysis of pAkt, Akt and PI3K, the representative results. ( e ) pp44/42 and p44/42 protein expression: Western Blot analysis of pp44/42 and p44/42, the representative results. p < 0.05 was considered statistically significant. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 as compared to the control. AOH alternariol. GPER1 G protein-coupled estrogen receptor 1. G15 selective GPER1 receptor antagonist. G1 selective GPER1 agonist, Cnt control (non-treated cells), Vin vinculin, HIF1α hypoxia-inducible factor 1-alpha, CLDN1 claudin 1, CLDN3 claudin 3, CLDN4 claudin 4 , p44/42 extracellular signal-regulated kinases, phospho-p44/42 phospho-extracellular signal-regulated kinases, Akt protein kinase B, pAkt phospho-protein kinase B, PI3K phosphoinositide 3-kinase.

Article Snippet: Akt (#9272), phospho-Akt (Ser473) (#4060), PARP1 (#46,011), SOD1 (#4266), SOD2 (#13,141), pH2AX (#9718), CLDN1 (#13,995), CLDN3 (#83,609), PI3K (#11,889), p44/42 (#4695), phospho-p44/42 (#4370), CLDN4 (STJ114778), GPER1 (STJ192629) (St John’s Laboratory Ltd, University Way, London) antibodies were used according to the manufacturer’s recommendations.

Techniques: Expressing, Gene Expression, Western Blot, Control

Journal: PLoS ONE

Article Title: Plasma membrane expression of G protein-coupled estrogen receptor (GPER)/G protein-coupled receptor 30 (GPR30) is associated with worse outcome in metachronous contralateral breast cancer

doi: 10.1371/journal.pone.0231786

Figure Lengend Snippet: A and B , images of GPR30 staining intensity level 3 without plasma-membrane staining (GPR30 PM -) ( A ) and with plasma-membrane staining (GPR30 PM +) ( B ). C - G , images of GPR30 total staining (GPR30 TOT ) as negative ( C ), very weak (level 1) ( D ), weak (level 2) ( E ), moderate (level 3) ( F ) and very strong (level 4) ( G ). Bar , 50 μm.

Article Snippet: The specificity of the polyclonal goat GPR30 antibody (AF 5534; R&D Systems) was analyzed in MCF7 human breast cancer cells (American Type Culture Collection), a cell line used extensively to study native GPR30 [ ].

Techniques: Staining, Clinical Proteomics, Membrane

Journal: PLoS ONE

Article Title: Plasma membrane expression of G protein-coupled estrogen receptor (GPER)/G protein-coupled receptor 30 (GPR30) is associated with worse outcome in metachronous contralateral breast cancer

doi: 10.1371/journal.pone.0231786

Figure Lengend Snippet: Association between total GPR30 intensity score (GPR30 TOT ), plasma membrane GPR30 (GPR30 PM ) status, and various clinicopathological variables.

Article Snippet: The specificity of the polyclonal goat GPR30 antibody (AF 5534; R&D Systems) was analyzed in MCF7 human breast cancer cells (American Type Culture Collection), a cell line used extensively to study native GPR30 [ ].

Techniques: Clinical Proteomics, Membrane, Biomarker Discovery, Staining

Journal: PLoS ONE

Article Title: Plasma membrane expression of G protein-coupled estrogen receptor (GPER)/G protein-coupled receptor 30 (GPR30) is associated with worse outcome in metachronous contralateral breast cancer

doi: 10.1371/journal.pone.0231786

Figure Lengend Snippet: The change in GPR30 intensity (level 0–4) between paired BC and LGL. In relation to the tumor assumed to have developed earlier, the intensity shift in the second tumor is characterized as decreasing, stable or increasing for each tumor pair respectively. The intensity shift was assessed statistically using Wilcoxon matched pairs signed rank test.

Article Snippet: The specificity of the polyclonal goat GPR30 antibody (AF 5534; R&D Systems) was analyzed in MCF7 human breast cancer cells (American Type Culture Collection), a cell line used extensively to study native GPR30 [ ].

Techniques:

Journal: PLoS ONE

Article Title: Plasma membrane expression of G protein-coupled estrogen receptor (GPER)/G protein-coupled receptor 30 (GPR30) is associated with worse outcome in metachronous contralateral breast cancer

doi: 10.1371/journal.pone.0231786

Figure Lengend Snippet: Prognostic effect of total GPR30 staining (GPR30 TOT ) and PM-specific GPR30 (GPR30 PM ) staining of BC2 and LGL2 calculated by Cox proportional hazards model with Wald test.

Article Snippet: The specificity of the polyclonal goat GPR30 antibody (AF 5534; R&D Systems) was analyzed in MCF7 human breast cancer cells (American Type Culture Collection), a cell line used extensively to study native GPR30 [ ].

Techniques: Staining

Journal: PLoS ONE

Article Title: Plasma membrane expression of G protein-coupled estrogen receptor (GPER)/G protein-coupled receptor 30 (GPR30) is associated with worse outcome in metachronous contralateral breast cancer

doi: 10.1371/journal.pone.0231786

Figure Lengend Snippet: A , cumulative incidence of BCD in relation to GPR30 PM staining of BC2. B , cumulative incidence of BCD in relation to GPR30 PM staining of LGL2. In A and B , cumulative incidences of competing event (death from other cause than BC) are shown for comparison.

Article Snippet: The specificity of the polyclonal goat GPR30 antibody (AF 5534; R&D Systems) was analyzed in MCF7 human breast cancer cells (American Type Culture Collection), a cell line used extensively to study native GPR30 [ ].

Techniques: Staining, Comparison

Journal: PLoS ONE

Article Title: Plasma membrane expression of G protein-coupled estrogen receptor (GPER)/G protein-coupled receptor 30 (GPR30) is associated with worse outcome in metachronous contralateral breast cancer

doi: 10.1371/journal.pone.0231786

Figure Lengend Snippet: A , cumulative incidence of BCD in relation to GPR30 TOT of BC2 in the whole cohort. B , cumulative incidence of BCD in relation to the five GPR30 staining intensity levels of BC2. C , cumulative incidence of BCD in relation to GPR30 TOT of LGL2. In A - C , cumulative incidences of competing event (death from other cause than BC) are shown for comparison.

Article Snippet: The specificity of the polyclonal goat GPR30 antibody (AF 5534; R&D Systems) was analyzed in MCF7 human breast cancer cells (American Type Culture Collection), a cell line used extensively to study native GPR30 [ ].

Techniques: Staining, Comparison

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Shikonin Mediates Apoptosis through G Protein-Coupled Estrogen Receptor of Ovarian Cancer Cells

doi: 10.1155/2022/6517732

Figure Lengend Snippet: Regulation of EGFR/PI3K/p-AKT signaling pathway mediated by SK via GPER in SKOV3 and A2780 cells. (a) The expression of EGFR, p-EGFR, PI3K, AKT and p-AKT following G-1 and G-15 treatment together with 8.6 μ M SK in SKOV3 cells. (b) The expression of EGFR, p-EGFR, PI3K, AKT, and p-AKT following G-1 and G-15 treatment together with 0.56 μ M SK in A2780 cells. The results are means of three independent replicates ± S.D. ∗∗ P < 0.01 or ∗ P < 0.05 vs. control group, △△ P < 0.01 or △ P < 0.05 vs. SK treatment group were considered as statistically significant.

Article Snippet: GPER-specific agonists G-1 and G-15 were obtained from MCE (Shanghai, China).

Techniques: Expressing, Control

Journal: Endocrine Disruptors

Article Title: Rapid, nongenomic signaling effects of several xenoestrogens involved in early- vs. late-stage prostate cancer cell proliferation

doi: 10.4161/23273747.2014.995003

Figure Lengend Snippet: Figure 2. ER subtype (a, b, and GPR30) levels (total vs. membrane) in LAPC-4 and PC-3 prostate cancer cells. The negative control samples used no primary antibody (Ab) for any of the ER subtypes, as indicated by the first bar and the shaded bar extending horizontally across the graph (average §SEM ). *denotes significance from controls at P < 0.05.

Article Snippet: ER Abs used included ERa (MC-20, Santa Cruz Biotechnology, Dallas, TX); ERb (clone 9.88, SigmaAldrich); and GPR30 (Cat. No. NLS4271, Novus Biologicals, Littleton, CO).

Techniques: Membrane, Negative Control

Journal: Endocrine Disruptors

Article Title: Rapid, nongenomic signaling effects of several xenoestrogens involved in early- vs. late-stage prostate cancer cell proliferation

doi: 10.4161/23273747.2014.995003

Figure Lengend Snippet: Figure 4. ROS levels after treatment with 10¡10M E2, 10¡6MH2O2, 10¡9MBPA, 10¡7M coumestrol, 10¡7M genistein, and 10¡8M resveratrol, § ER subtype-selective antagonists. Antagonists (Antag) were 10¡7M MMP for ERa; 10¡6M PHTPP for ERb; and 10¡6M G15 for GPR30. ROS levels were mea- sured after 15 min of each XE treatment (the optimal response time). *denotes significance from vehi- cle (V) controls at P < 0.05, while # denotes significance from paired XE treatment values (P < 0.05). ERa inhibition was significantly different vs. resveratrol alone in PC-3 cells ($) at P < 0.09. The shaded horizontal bars represent the response to vehicle (V) § SEM.

Article Snippet: ER Abs used included ERa (MC-20, Santa Cruz Biotechnology, Dallas, TX); ERb (clone 9.88, SigmaAldrich); and GPR30 (Cat. No. NLS4271, Novus Biologicals, Littleton, CO).

Techniques: Inhibition

Journal: Endocrine Disruptors

Article Title: Rapid, nongenomic signaling effects of several xenoestrogens involved in early- vs. late-stage prostate cancer cell proliferation

doi: 10.4161/23273747.2014.995003

Figure Lengend Snippet: Figure 5. Cyclin D1 phosphorylation and degradation by XEs, and inhibition by ER-selective antago- nists. Cyclin phosphorylation was measured at 1–60 min, and total cyclin D1 levels over 16 h of XE treatment. For 5A and 5B, LAPC-4 and PC-3 cells were pretreated with antagonists (Antag) for each of the 3 ER subtypes: a (MPP), b (PHTPP), and GPR30 (G15,) and then treated with 10¡9M BPA, 10¡7M coumestrol, 10¡7M genistein, 10¡8M resveratrol, 10¡10M E2 or 10¡6M DES. Shaded horizontal bars rep- resent V § SEM. * denotes significance compared to vehicle (V) at P < 0.05. # denotes significance from paired XE treatment responses at P < 0.05. For 5C and 5D, LAPC-4 and PC-3 cells were treated with each XE for the times indicated and total cyclin D1 levels were measured with a plate immunoassay.

Article Snippet: ER Abs used included ERa (MC-20, Santa Cruz Biotechnology, Dallas, TX); ERb (clone 9.88, SigmaAldrich); and GPR30 (Cat. No. NLS4271, Novus Biologicals, Littleton, CO).

Techniques: Phospho-proteomics, Inhibition

Journal: Animals : an Open Access Journal from MDPI

Article Title: Development of Porcine Accessory Sex Glands

doi: 10.3390/ani14030462

Figure Lengend Snippet: List of antibodies used for immunohistochemical labeling.

Article Snippet: GPER , Aviva Systems Biology , ARP62244 , 1:100 , Overnight, 4 °C, TBS , ImmPACT Red ® , [ ] .

Techniques: Immunohistochemical staining, Labeling