Estradiol/GPER affects the integrity of mammary duct-like structures in vitro

High estrogen concentration leads to an inflammatory reaction in the mammary gland tissue in vivo; however, the detailed mechanism underlying its specific effects on the breast duct has not been fully clarified. We used 3D-cultured MCF-10A acini as a breast duct model and demonstrated various deleterious effects of 17-β estradiol (E2), including the destruction of the basement membrane surrounding the acini, abnormal adhesion between cells, and cell death via apoptosis and pyroptosis. Moreover, we clarified the mechanism underlying these phenomena: E2 binds to GPER in MCF-10A cells and stimulates matrix metalloproteinase 3 (MMP-3) and interleukin-1β (IL-1β) secretion via JNK and p38 MAPK signaling pathways. IL-1β activates the IL-1R1 signaling pathway and induces continuous MMP-3 and IL-1β secretion. Collectively, our novel findings reveal an important molecular mechanism underlying the effects of E2 on the integrity of duct-like structures in vitro. Thus, E2 may act as a trigger for ductal carcinoma transition in situ.

duct-like structures (Fig. 1a). In normal breast tissue, the centrosomes were located inside the breast duct and showed the same polarity as the 3D model ( Supplementary Fig. 1b).
In the 3D model treated with 32 nM E2, increased partial disruption of cell-cell adhesion and basement membrane were observed compared with those in control (Fig. 1a,b). However, the disruption was not observed when treated with 32 nM 17α-estradiol, which shows no specific binding activity for GPER. And E2-Glow treatment (32 nM) disrupted the basement membrane as well as did E2 (Supplementary Fig. 2e).
In particular, p53-knockout MCF-10A cells accumulated without anoikis in the ducts, and some of the cells inside the duct were released out of the basement membrane following E2 treatment ( Supplementary Fig. 1c). To better visualize the basement membrane of the glandular model, we employed SEM next. Partial basement membrane loss was observed following E2 treatment, and microvilli on the cell surface constituting the duct were detected in the gap (Fig. 1c).
Estrogens bind to the estrogen receptor and GPER 7,8 . MCF-10A cells do not express ERα but express GPER (Fig. 1d). GPER was expressed in mammary gland tissues in normal breast ducts, ductal carcinoma in situ (DCIS), and invasive ductal carcinoma (IDC) in immunofluorescence staining (Fig. 1e). To investigate the potential effects of estradiol on cells via GPER, E2-Glow-fluorescently labeled E2-was added to MCF-10A cells. Immunostaining confirmed that E2-Glow was colocalized with GPER ( Fig. 1f). Furthermore, we performed E2-Glow and GPER binding experiments. E2-Glow and FLAG-GPER were reacted and immunoprecipitated with an anti-FLAG antibody. Fluorescence of the sedimentation product increased with E2-Glow concentration (Fig. 1g).
Indeed, cAMP activity significantly increased at 15 and 30 min after E2 addition but showed no activity after 24 h. The p38, JNK, and IkB signaling pathways have been implicated in cAMP signaling 22,23 . To identify the signaling cascades involved in these E2-induced pathways, we examined the phosphorylation of molecules within the p38, JNK, and IkB pathways. p38, JNK, and IkB were rapidly phosphorylated following E2 stimulation, reaching a peak at 15 min and declining to the basal levels in 30 min (Fig. 2b,d,f). E2-Glow stimulation increased p38 phosphorylation, although 17α-estradiol did not induce p38 phosphorylation ( Supplementary Fig. 2d). c-Jun (on Ser63), a p38 and JNK substrate, was phosphorylated to an extent similar to p38 and JNK (Fig. 2g). Moreover, we confirmed the association between GPER and the p38/JNK signaling pathway. Incubating cells with a GPER antagonist(G-15) reduced the E2-induced increases in p38 and JNK phosphorylation (Fig. 2c,e). To validate the role of GPER in E2 stimulation, we next used siRNAs to knock down GPER ( Supplementary Fig. 2f-h). In control siRNA-transfected MCF-10A cells, E2 stimulation markedly increased p38 and JNK phosphorylation. However, in cells transfected with siRNA-GPER, E2-induced phosphorylation declined. We used Accell siRNA to investigate whether E2 and E2-Glow disrupted the basement membrane of 3D-cultured (>7 days) GPER-knockdown cells. And there was no basement membrane collapse observed in the Accell siRNA-GPER group as opposed to that in the siRNA-control group following E2 treatment (Fig. 2h).
Furthermore, we used MCF-7 cells expressing both ERα and GPER to examine whether E2 stimulation leads to p38 and JNK phosphorylation to a similar extent as that observed in MCF-10A cells. Phosphorylation slightly increased up to 60 min following E2 exposure ( Supplementary Fig. 2i,j). These results indicated that E2-dependent p38 and JNK activation occurs via GPER.
Estradiol promotes MMP-3 secretion by MCF-10A cells, and basement membrane disruption is rescued by an MMP-3 inhibitor. E2 stimulation disrupted cell-cell adhesion (cadherin) and the basement membrane (laminin V) in the 3D MCF-10A model (Fig. 1a). Cadherin and laminin V are the targets of MMP-3-a member of the MMP family of extracellular proteases 24,25 . Furthermore, MMP-3 is a target gene of the transcription factor AP-1, which is located downstream of JNK and p38 [26][27][28][29] . Therefore, we considered that E2 may induce MMP-3 secretion. To determine the involvement of MMP-3 in the collapse of cell-cell adhesion and basement membrane following E2 stimulation, we tested the effects of E2 on pro-MMP-3 expression in cultured MCF-10A cells. Cells were treated with E2 (32 nM) for 24 and 48 h, and pro-MMP-3 expression was analyzed via western blotting (Fig. 3a). Pro-MMP-3 expression was confirmed 24 h after E2 addition. To measure the activity of MMP-3 secreted into the medium, cells were treated with E2 for 24 and 48 h, and the media was analyzed using the MMP-3 Activity Assay Kit. The activity of secreted MMP-3 extracellularly increased in a time-dependent manner (Fig. 3b), and was suppressed following the addition of N-isobutyl-N-(4-methoxyphenylsulfonyl)-glycyl hydroxamic acid (NNGH)-a commonly used semi-selective MMP-3 inhibitor (Fig. 3c). To evaluate whether E2-induced MMP-3 secretion was responsible for the disruption of the intercellular junctions in confluent MCF-10A cells, we used immunofluorescence analysis to determine cadherin localization (Fig. 3d). And consistent with our observations in MCF-10A cells, NNGH and E2 combination could rescue adherent junctions in a concentration-dependent manner. Furthermore, cadherin degradation in siRNA-pro-MMP-3-treated MCF-10A cells was clearly suppressed compared with that in siRNA-control cells. These results suggested that E2 induces MMP-3 secretion and acts on cadherin ( Supplementary Fig. 3a). Then, we investigated the effects of NNGH on basement membrane using the 3D model (Fig. 3e). Cadherin and basement membrane disappearance due to E2 exposure was restored to normal levels (E2 0 nM) by NNGH. These results suggested that the E2-induced MMP-3 secretion led to the loss of cell-cell adhesion and basement membrane in 3D model.   www.nature.com/scientificreports www.nature.com/scientificreports/ Estradiol induces MCF-10A cells pyroptosis. IL-1β is secreted by fibroblasts expressing GPER via E2 30,31 . Therefore, we next examined whether MCF-10A cells exposed to E2 secreted IL-1β into the medium (Fig. 4a). IL-1β secretion showed the highest level following 32 nM E2 addition to MCF-10A cells for 60 h. IL-1β secretion in the medium increased with time and was detected for up to 120 h (Fig. 4b, Supplementary Fig. 3b). IL-1β is expressed as pro-IL-1β by various transcription factors, including AP-1 and NFκB 32   www.nature.com/scientificreports www.nature.com/scientificreports/ then processed by caspase-1 activated via inflammasome to become mature IL-1β [35][36][37] . Caspase-1 activation via inflammasome is induced by reactive oxygen species (ROS) production 38,39 . Therefore, we investigated whether E2 induces ROS production and caspase-1 activation. Fluorescence-labeled E2 and MitoTracker probe for labeling live cell mitochondria were added to MCF-10A cells, and the cells were observed after 20 min. E2 accumulation in the mitochondria was confirmed (Fig. 4c). Moreover, ROS production was detected at 15 min following exposure to E2 (32 nM), and was reduced after the addition of a negative control antioxidant (N-acetyl cysteine) (Fig. 4d). The experimental system functioned normally under increased ROS production after the addition of positive control (antimycin A). Mitochondrial ROS activate caspase-1 via the NLRP3 inflammasome 40,41 . In the current study, E2 (2 and 32 nM) activated caspase-1 (Fig. 4e, Supplementary Fig. 3c), and the activity was suppressed by the caspase-1-specific inhibitor Ac-YVAD-CHO.
Activated caspase-1 cleaves GSDMD, which then forms a complex and opens a membrane hole in the cell membrane [42][43][44][45] . IL-1β is then secreted out of the cell through those membrane pores 46 . Our results showed that E2 (32 nM) converted wild-type FLAG-GSDMD in MCF-10A cells to cleaved FLAG-GSDMS in 1 h (Fig. 4f). The cleavage product matched the size of the N-terminal fragment in cells. Then we confirmed that endogenous GSDMD was cleaved similar to FLAG-GSDMD in E2-stimulated MCF-10A cells (Fig. 4g). We next used confocal immunofluorescence microscopy to visualize the cellular distribution of endogenous GSDMD using an anti-N-terminal GSDMD recognition antibody following E2 stimulation. The cleaved N-terminal GSDMD was localized on the cell membranes which not subjected to membrane permeabilization (Fig. 4h). The presence of morphological structures, termed pyroptotic bodies, in cells undergoing E2 stimulation has been reported 47 . Pyroptotic bodies show a similar diameter (1-5 μm) to apoptotic bodies. SEM and phase-contrast microscopy revealed that E2-treated MCF-10A cells progressed to form protrusions of similar sizes as pyroptotic bodies (Fig. 4i, Supplementary Fig. 3d). These results confirmed that E2-treated MCF-10A cells secreted IL-1β and undergo pyroptosis.
E2 stimulation led to IL-1β secretion in MCF-10A cells (Fig. 4a,b). Next, we examined whether IL-1β secretion by E2-treated MCF-10A cells was directly related to IL-1R expression. To this end, IL-1R protein levels in MCF-10A cells were examined for the presence of fractionated cytoplasm and nuclei by western blotting (Fig. 5a). A549 cells were used as IL-1R-expressing controls and 293T cells as IL-1R-non-expressing controls. We confirmed that IL-1R was expressed in A549 and MCF-10A cells but not in 293T cells.
Estradiol induces MCF-10A cells apoptosis. IL-1β induces cellular apoptosis by releasing cytochrome c from mitochondria 48 . In the present study of E2 induced pyroptosis, we observed that E2 was accumulated in the mitochondria, and ROS production was detected (Fig. 4c,d). E2 localizes directly to mitochondria without via GPER. Actually, when GPER was knocked down using siRNA, E2-Glow was localized in mitochondria (Fig. 4c). ROS production is an important factor inducing apoptosis. These results suggested that E2 induced apoptosis in addition to pyroptosis. In fact, E2 elicited several responses, including the release of cytochrome c; activation of caspase-3; and an increase in the number of annexin V-positive cells, sub-G1 cells and apoptotic cells (Fig. 6a-f). Furthermore, E2 activated caspase-3 in MF10A cells that constituted the breast duct model (Fig. 6g). And in normal breast tissues, DCIS, and IDC tissues, we examined whether pyroptosis and apoptosis occurred in the same cells which form the breast duct. To this end, tissue arrays were subjected to immunostaining with the anti-caspase-1 antibody-a marker for pyroptosis-and the anti-caspase-3 antibody-a marker for apoptosis. Caspase-1 and caspase-3 activation were observed in the same cells in both DCIS and IDC (Fig. 6h,i). These data are consistent with our hypothesis that caspase-1 and caspase-3 function as downstream components of E2-stimulated pyroptosis and apoptosis signaling pathways.   www.nature.com/scientificreports www.nature.com/scientificreports/

Discussion
In this study, we examined the involvement of E2 in breast duct collapse. In particular, we focused on the mechanism through which E2 stimulation leads to IL-1β and MMP-3 secretion. For this purpose, we constructed a breast duct model using MCF-10A cells and evaluated the effect of E2 on this issue (Fig. 7). E2 binding to GPER led to cAMP activation and MMP-3 and IL-1β secretion. MMP-3 further degraded cadherin, which adheres to cells that make up the ducts, and laminin in the basement membrane. Moreover, E2 activated caspase-1, which degraded GSDMD to induce pyroptosis. Caspase-1 activation requires inflammasome formation, which is induced by ROS production. In this study, we also confirmed ROS production by E2. Furthermore, E2 accumulation in the mitochondria induced apoptosis along with cytochrome c release and caspase-3 activation. In contrast, IL-1β binds to IL-1R and activates various intracellular signals to promote MMP-3 secretion and induce apoptosis [49][50][51] . In fact, IL-1β stimulation induced MMP-3 secretion (Fig. 5g). Based on these results, E2 may trigger sequential ductal structure disruption via its synergistic action with IL-1β.
As a result of the genomic actions of E2 and GPER, IL-1β and MMP-3 were produced (Figs. 3b and 4a). IL-1β is elevated in various types of cancers, and IL-1β-producing tumors showed a worse prognosis in the Human Protein Atlas. IL-1β was expressed at very low levels in normal mammary epithelial cells; however, its expression was significantly elevated in DCIS and IDC 52,53 . Meanwhile, the mechanism underlying this process remains unclear. E2 concentrations in mammary gland tissues also increase with tumor progression (DCIS and IDC) 54 . Our results indicated that IL-1β secretion was significantly dependent on E2 concentration (Fig. 4a). Therefore, E2 concentration might increase with DCIS and IDC progression, thus promoting IL-1β production and eventually disrupting the ductal structure.
In this study, we showed that estrogen (32 nM) disrupted the basement membrane and ductal structure. E2 concentration in malignant breast tissue is ~1-2 pmol/g tissue 55 . If water content of the tissue is estimated to be ~60%, E2 concentration would be ~1.7-3.4 nM. As such, E2 concentration used in this study was ~10-20 times higher than the estimated E2 concentration in tissues. This poses a question of whether E2 at a concentration of 32 nM confers physiological effects. While there is no definitive answer to this question, E2 metabolites (E1, E2, and E3) are present in tissues 56,57 . For example, blood E3 concentrations of nonpregnant women are 3-19 times higher than the combined E1 and E2 concentrations 58 . Furthermore, E1 and E3 are closely involved in nongenomic GPER signaling pathways, suggesting that their role as are GPER agonists 59,60 . Therefore, comparing E2 alone in a system is not accurate. During pregnancy, blood E2 concentration increases to ≥100 nM; thus, while 32 nM E2 is considered relatively high, it is within the range of physiological conditions. E2 binds to both GPER and ERα receptors, so how do cells presenting both receptors, such as MCF-7 cells, use each receptor properly? Because of E2 exhibits two functions (nongenomic and genomic) and E2-GPER signal transduction occurs earlier than E2-ERα signal transduction, the receptors are presumably used at different times. In MCF-10A cells (ERα − , GPER + ), cAMP activation increased in 15 min following E2 addition and decreased after 30 min. After 24 h, the activity was not recognized (Fig. 2a). In addition, p38 and JNK, which are downstream of cAMP, were also phosphorylated within 30 min (Fig. 2b,d). As a nongenomic effect of E2 on MCF-7 cells, p38 and JNK phosphorylation was detected within 60 min after E2 addition ( Supplementary  Fig. 2i,j). In contrast, as a genomic action of E2 on MCF-7 cells, the transcriptional target gene pS2 of E2-ERα was expressed 24 h following E2 addition (data not shown). Thus, in cell types expressing both receptors, E2 appears to work synergistically via nongenomic and genomic actions.
In conclusion, by using a duct-like model, we clarified the mechanism through which estrogen actions destroy the ductal structure in breast cancer. Breast duct collapse may initiate breast cancer invasion, leading to cancer cell release in the breast duct as cancer progresses.
SiRNA treatment. siRNA oligonucleotides for human GPER (siRNA ID: s6054) and human MMP-3 (siRNA ID: s8854) used in this study were purchased from Ambion. The nonspecific negative control was purchased from Dharmacon (siRNA: D-001810-01-50). All SiRNA treatments were performed using the Lipofectamine RNAiMAX Reagent (Invitrogen) according to the manufacturer's instructions.

Plasmids and transfection.
To obtain the expression vector coding for human active-type IL-1β with an N-terminal GST tag, the gene was amplified by PCR and cloned into the pGEX 4T-2 plasmid (Amersham Biosciences, Piscataway, NJ, USA). The gene was then amplified by PCR using the following primers: 5′-TATGGATCCCCAGGAATTCTCGCACCT-3′ and 5′-GCGCTCGAGTTAGGAAGACACAAATTG-3′. PCR amplification was performed using 40 cycles at 94 °C for 10 s and 68 °C for 30 s. The IL-1β gene was inserted between the BamH1 and Xho1 sites of the pGEX 4T-2 plasmid, and the plasmid was verified by DNA sequencing and protein expression. Further confirmation was performed by western blotting and IL-1β ELISA. To obtain the expression vector coding for human GSDMD with an N-terminal FLAG tag, the gene (human liver) was amplified by PCR and cloned into the p3XFLAG-Myc-CMV plasmid vector (Sigma). The gene was amplified by PCR using the following primers: 5′-TTGCGGCCGCGAATTCAATGGGGTCGGCCTTTGAG-3′ and 5′-TCGACTGGTACCGATATCATGTGGGGCTCCTGGCTCAG-3′. PCR amplification was performed using 40 cycles at 98 °C for 10 s and 68 °C for 60 s. The Gasdermin-D gene was inserted between the EcoR I and EcoR V sites of the p3XFLAG-Myc-CMV plasmid, and the plasmid was verified by DNA sequencing. All transfections were performed using the Lipofectamine TM 3000 Transfection Kit (Invitrogen), according to the manufacturer's instructions.

Bacterial expression of recombinant proteins. Recombinant GST-tagged IL-1β was overexpressed in
E. coli BL21 (DE3) physS-competent cells using the following protocol. Briefly, cells were grown in LB medium supplemented with 100 mg/mL ampicillin at 37 °C until an OD 600 of 0.6-0.8 was obtained. Protein expression was induced with 0.5 mM isopropyl β-D-thiogalactoside. Cells were further cultured at 37 °C for 2 h and harvested by centrifugation. For purification, cell pellets were resuspended in lysis buffer containing 50 mM Tris-HCl (pH 7.4), 400 mM NaCl, 0.5 mM EDTA, 5 mM MgCl 2 , 5% glycerol, and 1 mM DTT supplemented with 1 mg/ml lysozyme and 0.5 mM PMSF. Cells were lysed by sonication, and the lysates were clarified by centrifugation. The supernatants were then applied to a GST microspin column (cat. 28-9523-59, GE Healthcare). Proteins were eluted using 20 mM reduced glutathione (pH 8.0), and the eluted fractions were then dialyzed against phosphate-buffered saline (PBS). cAMP assays. MCF-10A cells were cultured in 96-well white, clear bottom plates at a density of 8 × 10 3 cells/ well. After 2 days of incubation, cells were treated with 32 nM E2 for 15 min, 30 min, 24 h, and 48 h. Following E2 treatment, the cyclic adenosine monophosphate (cAMP) assay was performed according the manufacturer's instructions for cAMP-Glo Assay (V1501: Promega). This is a bioluminescent assay for monitoring changes in intracellular cAMP concentrations. In the assay, cells were lysed to release cAMP, followed by the addition of the cAMP Detection Solution containing protein kinase A (PKA) and a kinase substrate. The Kinase-Glo Reagent was added next to terminate the PKA reaction and detect the remaining ATP via a luciferase reaction. The plates were then read using a microplate reader (EnSpire; PerkinElmer). Luminescence was correlated to cAMP concentration using a cAMP standard curve.
Quantitative IL-1β detection. The human IL-1β ELISA Kit (BMS224/2: Invitrogen), an enzyme-linked immunosorbent assay, was used for the quantitative detection of human IL-1β. MCF-10A cells were cultured in 96-well clear plates at a density of ~ 4 × 10 3 cells/well. After 1-2 days of incubation, the medium was changed and replaced with a fresh medium containing 32 nM E2. This treatment lasted for 60, 72, 96, and 120 h. Following treatment, the supernatant was transferred to a microwell plate coated with a monoclonal antibody against human IL-1β, which the kit provided. The human IL-1β ELISA assay was performed according to the provided protocol.
MMP-3 activity assay. The MMP-3 Activity Assay Kit (ab118972: Abcam) was used to measure MMP-3 activity in cell culture media. MCF-10A cells were cultured in 96-well clear plates at a density of ~ 8 × 10 3 cells/ well for 1 day. After that, the cells were treated with 2 or 32 nM E2 for 24 or 48 h. To directly measure MMP-3 activity, cells were transferred to a 96-well black plate. After reacting with the MMP-3 substrate (prepared according to the protocol), the plate was read at Ex/Em = 325/393 nm twice in 2 h.

ROS detection cell-based assay. The ROS Detection Cell-Based Assay Kit (cat. no. 601290: Cayman
Chemical) was used to detect ROS production. MCF-10A cells were plated at a density of 8 × 10 3 cells/well in black tissue-treated 96-well plates and cultured for 2 days. Then, 32 nM E2 was added for 15 and 30 min. Fluorescence was measured at an excitation wavelength of 480 nm and an emission wavelength of 570 nm.
Immunofluorescence microscopy. Cells were washed in PBS; fixed in 3.4% formaldehyde in PBS for 10 min on ice; and sequentially permeabilized in 50%, 75%, and 95% ethanol on ice for 5 min each. Cells were blocked with donkey serum for 30 min at room temperature, followed by incubation with a primary antibody for 1 h at room temperature. Cells were then washed three times with PBS for 5 min each and incubated with Alexa-488-or Alexa-594-conjugated secondary antibody for 30 min at 37 °C. After washing twice with PBS, DNA was stained with 1 μg/mL bisbenzimide (Hoechst 33258). Samples were then examined under a TCS SP8 confocal microscope (Leica Microsystems).
Immunohistochemical staining. Tissue arrays (HBreD060CS05 and BR486) were purchased from US Biomax. After deparaffinization and rehydration, antigen retrieval treatment was carried out in an autoclave at 121 °C for 20 min in antigen activation buffer (pH 9.0) (Nichirei Corp.). Tissue arrays were blocked in donkey serum for 30 min at room temperature, followed by incubation with primary antibody for 1 h at room temperature. Cells were then washed three times with PBS for 5 min each and incubated with Alexa-488 or Alexa-594 conjugated secondary antibodies for 30 min at 37 °C. After washing twice with PBS, DNA was stained with 1 μg/ mL bisbenzimide (Hoechst 33258).
Immunoblotting. MCF-10A cells were harvested and washed in PBS. After centrifugation, the cell pellet was suspended in sonication buffer (20 mM Tris-HCl (pH 8.0), 100 mM NaCl, and 1 mM EDTA) containing protease inhibitors (completely EDTA-free; Roche). For phosphate protein detection, the buffer also contained a phosphatase inhibitor cocktail (50 mM sodium fluoride, 10 mM β-glycerophosphate, 10 mM sodium pyrophosphate, and 1 mM activated sodium orthovanadate; Calbiochem). The cells were then lysed and centrifuged at 15000 × g for 30 min at 4 °C. The retrieved proteins were resolved by SDS-PAGE and transferred onto a PVDF membrane (Millipore). The membrane was then incubated with primary antibodies for 1 h after blocking in SuperBlock Blocking Buffer (Thermo Scientific) at room temperature. The membrane was then probed with secondary antibodies for 1 h at room temperature. Blots were developed using the Amersham TM ECL Select TM Western Blotting Detection Reagent (GE Healthcare) and exposed to ImageQuant LAS 500, according to the manufacturer's instructions.
Three-dimensional cultures. Matrigel (40 μl) (Sigma-Aldrich) was added to each well of an eight-well glass chamber slide (Thermo Fisher) and placed in a 37 °C incubator to allow the Matrigel to solidify for 30 min. MCF-10A cells were diluted in MCF-10A medium to achieve a density of 25 × 10 3 cells/ml, and the cells were mixed with the medium containing 4% Matrigel at a 1:1 ratio. Next, 400 μl of this mixture was placed per well on top of the solidified Matrigel in each well of the chamber slide. The final overlay solution comprised 5 × 10 3 cells/ well in a medium containing 2% Matrigel. The cells were allowed to grow in a 5% CO 2 humidified incubator at 37 °C and were re-fed with MCF-10A medium containing 2% Matrigel every 4 days. Flow cytometry. A flow cytometer (FACSCalibur, BD Bioscience) was used for DNA cell analysis, cytochrome c expression, and annexin V analysis. The cell preparation for DNA cell analysis was performed as follows. Briefly, MCF-10A cells were washed with PBS and fixed in 70% ethanol. The fixed cells were then washed twice with PBS and treated with RNase A at 37 °C for 30 min. Finally, the cells were stained with propidium iodide and incubated in the dark for 30 min. Samples were analyzed by flow cytometry, and 10 4 cells were counted for each sample.

E2-glow binding assays. FLAG-GPER or FLAG was overexpressed in 293T cells and immunoprecipitated
with Dynabeads-FLAG antibody. FLAG-GPER or FLAG was bound to Dynabeads via a FLAG antibody. 10 uM E2-glow was reacted with the immunoprecipitated product at room temperature for 15 minutes. The plate was then washed three times with PBS, and the fluorescence value was measured using a microplate reader (EnSpire; PerkinElmer). A calibration curve was prepared in advance from the concentration of E2-Glow and the fluorescence value. The fluorescence value of E2-Glow reacted with FLAG was subtracted from the fluorescence value of E2-Glow reacted with FLAG-GPER, and the concentration of E2-Glow reacted with FLAG-GPER was calculated from the calibration curve.
Statistical analysis. The data in graphs are presented as mean ± SEM of three or more independent experiments and n values described in each figure legend represent each independent trial. Data for all experiments were analyzed using a Mann-Whitney U test. Values of P < 0.05 were considered statistically significant and the degree of significance is indicated in each figurelegend. *P < 0.05.