Accepted Manuscript Estrogen Activation of G protein-coupled Estrogen Receptor 1 Regulates Phosphoinositide 3-kinase and mTOR Signaling to Promote Liver Growth in Zebrafish and Proliferation of Human Hepatocytes Saireudee Chaturantabut, Arkadi Shwartz, Kimberley J. Evason, Andrew G. Cox, Kyle Labella, Arnout G. Schepers, Song Yang, Marianna Aravena, Yariv Houvras, Liliana Mancio-Silva, Shannon Romano, Daniel A. Gorelick, David E. Cohen, Leonard I. Zon, Sangeeta N. Bhatia, Trista E. North, Wolfram Goessling PII: S0016-5085(19)30031-9 DOI: https://doi.org/10.1053/j.gastro.2019.01.010 Reference: YGAST 62366 To appear in: Gastroenterology Accepted Date: 7 January 2019 Please cite this article as: Chaturantabut S, Shwartz A, Evason KJ, Cox AG, Labella K, Schepers AG, Yang S, Aravena M, Houvras Y, Mancio-Silva L, Romano S, Gorelick DA, Cohen DE, Zon LI, Bhatia SN, North TE, Goessling W, Estrogen Activation of G protein-coupled Estrogen Receptor 1 Regulates Phosphoinositide 3-kinase and mTOR Signaling to Promote Liver Growth in Zebrafish and Proliferation of Human Hepatocytes, Gastroenterology (2019), doi: https://doi.org/10.1053/j.gastro.2019.01.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Accepted Manuscript
Estrogen Activation of G protein-coupled Estrogen Receptor 1 RegulatesPhosphoinositide 3-kinase and mTOR Signaling to Promote Liver Growth inZebrafish and Proliferation of Human Hepatocytes
Saireudee Chaturantabut, Arkadi Shwartz, Kimberley J. Evason, Andrew G. Cox,Kyle Labella, Arnout G. Schepers, Song Yang, Marianna Aravena, Yariv Houvras,Liliana Mancio-Silva, Shannon Romano, Daniel A. Gorelick, David E. Cohen,Leonard I. Zon, Sangeeta N. Bhatia, Trista E. North, Wolfram Goessling
To appear in: GastroenterologyAccepted Date: 7 January 2019
Please cite this article as: Chaturantabut S, Shwartz A, Evason KJ, Cox AG, Labella K, Schepers AG,Yang S, Aravena M, Houvras Y, Mancio-Silva L, Romano S, Gorelick DA, Cohen DE, Zon LI, BhatiaSN, North TE, Goessling W, Estrogen Activation of G protein-coupled Estrogen Receptor 1 RegulatesPhosphoinositide 3-kinase and mTOR Signaling to Promote Liver Growth in Zebrafish and Proliferationof Human Hepatocytes, Gastroenterology (2019), doi: https://doi.org/10.1053/j.gastro.2019.01.010.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service toour customers we are providing this early version of the manuscript. The manuscript will undergocopyediting, typesetting, and review of the resulting proof before it is published in its final form. Pleasenote that during the production process errors may be discovered which could affect the content, and alllegal disclaimers that apply to the journal pertain.
Title: Estrogen Activation of G protein-coupled Estrogen Receptor 1 Regulates Phosphoinositide 3-kinase and mTOR Signaling to Promote Liver Growth in Zebrafish and Proliferation of Human Hepatocytes Short Title: GPER1 enhances liver growth Authors: Saireudee Chaturantabut1, Arkadi Shwartz1, Kimberley J. Evason2, Andrew G. Cox1,3, Kyle Labella1, Arnout G. Schepers4, Song Yang5, Marianna Aravena6, Yariv Houvras7, Liliana Mancio-Silva4, Shannon Romano8, Daniel A. Gorelick8, David E. Cohen6, Leonard I. Zon5,9,10,11, Sangeeta N. Bhatia4,12,13, Trista E. North5,10, Wolfram Goessling1,10,11,12,13,14. 1Genetics Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA. 2Department of Pathology, University of Utah, Salt Lake City, UT 84112, USA 3Peter MacCallum Cancer Centre, Melbourne, Victoria 3000, Australia.. 4Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA. 5Stem Cell Program, Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA 02115, USA. 6Division of Gastroenterology & Hepatology, Weill Cornell Medical College, New York, NY 10021, USA. 7Departments of Surgery and Medicine, Weill Cornell Medical College, New York, New York 10021, USA. 8Department of Pharmacology & Toxicology, University of Alabama at Birmingham, Birmingham, AL 35293, USA. 9 Howard Hughes Medical Institute, 4000 Jones Bridge Road, Chevy Chase, MA 20815, USA. 10Harvard Stem Cell Institute, Cambridge, MA 02138, USA. 11Dana-Farber Cancer Institute, Boston, MA 02215, USA. 12Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA, 02139, USA. 13Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. 14Divison of Gastroenterology, Massachusetts General Hospital, Boston, MA 02114 Grant Support: This study is supported by NIH NCI 5K08CA172288 (K.J.E.), R24OD017870 (L.I.Z.; W.G), R01DK090311, R01DK105198 (W.G.) and The Claudia Adams Barr Program in Cancer Research (W.G.). W.G. is a Pew Scholar in the Biomedical Sciences. Abbreviations: ANAS, anastrozole; ANT, adjacent normal tissue; Akt, protein kinase B; BPA, bisphenol A; BrdU, bromodeoxyuridine; CC, cholangiocarcinoma; DAPI, 4′,6-diamidino-2-phenylindole; DMBA, dimethylbenzanthracene; DMSO, dimethysulfoxide; EdU, ethynyldeoxyuridine; ERE, estrogen response element; ERK, extracellular signal-regulated kinases; E2, 17β-estradiol; ESR, estrogen receptor; FACS, fluorescence-activated cell sorting; ful, fulvestrant; GO, Gene Ontology; GPER1, G protein-coupled estrogen receptor 1; HCA, hepatocellular adenoma; HCC, hepatocellular carcinoma; HNF4α, hepatocyte nuclear factor 4α; hpf, hours post fertilization; hpT, hours post treatment; IACUC - Institutional Animal Care and Use Committee; IHC, immunohistochemistry; ISH, in situ hybridization; mTOR, mechanistic target of rapamycin; MO, morpholino antisense oligonucleotide; Mtz, metronidazole; PCNA, proliferating cell nuclear antigen; PI3K, phosphoinositide 3-kinase; qRT-PCR, quantitative reverse transcription polymerase chain reaction; TALEN, transcription activator-like effector
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nuclease; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; WT, wild-type. Correspondence: Address correspondence to Wolfram Goessling ([email protected]), Genetics Division, Brigham and Women’s Hospital, NRB458, 77 Ave Louis Pasteur, Boston, MA 02115, U.S.A, phone: 617-525-7710, fax: 617-525-4751. Disclosure: The authors have no conflict of interest to disclose. Transcript Profiling: Accession number in GEO is GSE92544.
Author Contributions: S.C. and W.G. conceived the study, designed experiments, and analyzed data. S.C. conducted all zebrafish experiments. A.S. performed confocal microscopy and Western Blots. K.J.E. acquired human tissues and performed pathological analysis of zebrafish and human liver tumors. A.G.C. analyzed metabolomics data. K.L. performed mRNA rescue, xenoestrogens, and long-term anastrozole zebrafish experiments. A.G.S. and L.M-S. performed primary human hepatocyte experiments. S.Y. and Y.H. analyzed RNA sequencing data. M.A. carried out liver cancer cell line experiments. S.R., D.A.G., D.E.C., L.I.Z., S.N.B, and T.E.N. provided overall input. S.C., T.E.N and W.G. wrote the manuscript. All authors reviewed and edited the manuscript. The authors thank Isaac Oderberg from the Goessling lab for creating the visual abstract, John Asara at the Metabolomics Core at the Beth Israel Deaconess Medical Center for assistance with the metabolomics experiments, and staff of Beth Israel Deaconess zebrafish core facility for fish husbandry.
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Title: Estrogen Activation of G Protein-coupled Estrogen Receptor 1 Regulates Phosphoinositide 3-kinase and mTOR Signaling to Promote Liver Growth in Zebrafish and Proliferation of Human Hepatocytes Abstract: Background & Aims: Patients with cirrhosis are at high risk for hepatocellular carcinoma (HCC) and often have increased serum levels of estrogen. It is not clear how estrogen promotes hepatic growth. We investigated the effects of estrogen on hepatocyte proliferation during zebrafish development, liver regeneration, and carcinogenesis. We also studied human hepatocytes and liver tissues. Methods: Zebrafish were exposed to selective modifiers of estrogen signaling at larval and adult stages; liver growth was assessed by gene expression, fluorescent imaging, and histologic analyses. We monitored liver regeneration after hepatocyte ablation and HCC development after administration of chemical carcinogens (dimethylbenzanthrazene). Proliferation of human hepatocytes was measured in a co-culture system. We measured levels of G protein-coupled estrogen receptor (GPER1) in HCC and non-tumor liver tissues from 68 patients by immunohistochemistry. Results: Exposure to 17 beta-estradiol (E2) increased proliferation of hepatocytes and liver volume and mass in larval and adult zebrafish. Chemical genetic and epistasis experiments showed that GPER1 mediates the effects of E2 via the PI3K–Akt–mTOR pathway: gper1-knockout and mtor-knockout zebrafish did not increase liver growth in response to E2. HCC samples from patients had increased levels of GPER1, compared with non-tumor tissues; estrogen promoted proliferation of human primary hepatocytes. Estrogen accelerated hepatocarcinogenesis specifically in male zebrafish. Chemical inhibition or genetic loss of GPER1 significantly reduced tumor development in the zebrafish. Conclusions: In an analysis of zebrafish and human liver cells and tissues, we found GPER1 to be a hepatic estrogen sensor that regulates liver growth during development, regeneration, and tumorigenesis. Inhibitors of GPER1 might be developed for liver cancer prevention or treatment. KEY WORDS: sex hormone, signal transduction, transcription regulation, hepatocarcinogenesis
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Hepatocellular carcinoma (HCC) is the second most common cause of cancer mortality
worldwide, and the fastest-growing cause of cancer deaths in the U.S.1 Clinically relevant
biomarkers and therapies to detect and prevent HCC do not exist. Interestingly, chronic
liver disease and liver cancer are much more frequent in males,2 and men with cirrhosis
and HCC have elevated serum estrogen levels.3, 4 Further, patients undergoing surgical
liver resection display elevated serum levels of estrogen, suggesting the importance of
estrogenic regulation during liver regeneration.5 The mechanisms by which the liver
senses and responds to estrogen to impact liver growth and cancer formation remain
undetermined.
17β-estradiol (E2) is the most abundant biologically active form of estrogen. Canonical
estrogen signaling is mediated through nuclear hormone estrogen receptors 1
(ESR1/ERα) and 2 (ESR2/ERβ), resulting in transcriptional target gene activation. In
addition, E2 can exert non-canonical activity through the G protein-coupled estrogen
receptor 1 (GPER1, also known as GPR30). While roles of ESR1 and ESR2 have been
widely studied in the context of reproductive biology and cancer,6 the functional
consequences of GPER1 signaling are less well understood. Originally discovered in
breast cancer cells,7 GPER1 regulates proliferation and relaxation of vascular smooth
muscle.8 Several secondary messenger signals have been identified in different cellular
contexts, including Erk,7 PI3K,9 and Ca2+ release.10 GPER1 expression in hepatocytes,
and its potential role in hepatocyte proliferation and organ growth during development,
liver regeneration or cancer progression have not been previously characterized.
Here, we identify the essential function of E2 and GPER1 in the regulation of liver growth.
E2 induces cell cycle progression and increases hepatocyte proliferation and liver size in
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larval zebrafish. Surprisingly, these effects are not mediated through classic nuclear
hormone estrogen receptors, but via GPER1 and downstream activation of PI3K-mTOR
signaling. Importantly, GPER1 promotes sex-specific adult liver growth, and together with
mTOR is required for optimal liver regrowth after injury. In addition, GPER1 directly
modulates liver cancer formation: gper1-/- fish develop significantly fewer and smaller liver
tumors than wild-type siblings. The role of GPER1 is conserved in human liver, as liver
cancer tissue expresses increased GPER1, and primary human hepatocytes activate
AKT/mTOR and proliferate in response to E2. Chemical inhibition of GPER1 in vivo
significantly diminishes E2-induced tumor progression after chemical carcinogenesis,
particularly in males. We propose that GPER1 senses E2 to regulate PI3K/mTOR activity
and cellular proliferation during hepatic development and repair, and as such is an
important therapeutic target for liver cancer prevention and treatment.
Materials and Methods
Zebrafish husbandry
Zebrafish were maintained according to Institutional Animal Care and Use Committee
guidelines (IACUC) at Harvard Medical School.
Chemical Exposures
Zebrafish larvae were exposed to chemical modifiers, dissolved in 0.1% (v/v)
dimethylsulfoxide (DMSO), for 5hrs from 110-115 hours post fertilization (hpf) and
analyzed at 120hpf (unless otherwise specified).11 Adults were treated for 5-7hrs per
exposure. Chemicals are listed in Supplementary Table S5.
Morpholino Injection
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Morpholino oligonucleotides (MO) designed against esr1, esr2a, esr2b,11 gper1, and
mtor (GeneTools, Supplementary Table S3) and mismatched controls were injected into
one-cell stage embryos.
mRNA injection
Human GPER1 cDNA-containing plasmid (HsCD0032896) was transcribed using
mMESSAGE mMACHINE (Ambion). mRNA (200µg) was injected into 1-cell stage
embryos.
Generation of gper1 Mutants
TALENs targeting gper1 were obtained from Addgene (TAL3272, TAL3273).12 Adult
TALEN-injected fish (F0) were out-crossed to WT siblings, and progeny (F1) screened for
somatic mutations by Sanger sequencing (Supplementary Table S1). F1 mutants were
out-crossed for at least 4 generations to avoid possible off-target effects.
Western Blot Analysis
Pooled larvae (n=30-40) and cultured cells were processed for Western blot as previously
described.13 Antibodies are listed in Supplementary Table S4.
Whole Mount in situ Hybridization
Larvae were fixed and ISH performed according to standard protocols.14 gper1 probe was
generously gifted by David Volz.15
Liver Size Analysis
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ISH images were obtained by brightfield microscopy, and fabp10a:GFP reporter larvae
were imaged using fluorescence microscopy. 3D imaging of fabp10a:GFP fish was
performed with a Yokogawa W1 spinning disk confocal microscope. Image quantification
was achieved using ImageJ and Imaris software.
Histology and immunohistochemistry of zebrafish and human tissue
Zebrafish were fixed, sectioned and processed with hematoxylin/eosin (H&E) stains as
described16. For cell size analysis, zebrafish sections were stained with Pan-Cadherin
and 4′,6-diamidino-2-phenylindole (DAPI). Individual cell membrane staining was traced
and the cell area was measured using ImageJ. De-identified formalin-fixed paraffin-
embedded human liver sections were obtained at the University of Utah (IRB 00091019,
clinical characteristics summarized in Supplementary Table S6 and S7) or from
commercially available tissue arrays (OD-CT-DgLiv01-003, US Biomax, Supplementary
Table S7). IHC (antibodies in Supplementary Table S4) and terminal deoxynucleotidyl
transferase dUTP nick-end labeling (TUNEL) staining were performed using established
protocols.14 GPER1 staining was scored by a pathologist (K.J.E.).
Flow Cytometry and Cell Cycle Analysis
Fluorescence-activated cell sorting (FACS) on fabp10a:GFP larvae was performed as
previously described.14 Cell cycle analysis was achieved using the BrdU Flow kit (BD
Bioscience) and incubation in propidium iodide (PI) followed by flow cytometry analysis,
performed with FlowJo software.
qRT-PCR Analysis
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RNA was isolated from zebrafish larvae, processed and analyzed by qRT-PCR as
previously described.17 Primers are listed in Supplementary Table S2.
Chemical Carcinogenesis
Age-matched gper1-/- or wild-type fish were exposed to 5ppm dimethylbenzanthrazene
(DMBA) for 24hrs at 3, 4, and 5 weeks.18 Chemical exposures commenced at 6 weeks (3
times/week). Fish were monitored daily for survival and tumor formation until 33 weeks
post DMBA treatment. Fish were sacrificed upon reaching IACUC-approved tumor limits
or at 33 weeks post DMBA exposure. Histologic analysis was performed in a blinded
fashion by a board-certified pathologist with expertise in zebrafish liver histology (K.J.E.).
Steady-state Metabolomics Analysis
Adult livers were surgically removed and subjected to methanol extraction as previously
described.17, 19 Polar metabolites were identified using LC-MS/MS. Metabolic pathway
analysis was performed with MetaboAnalyst (http://www.metaboanalyst.ca).
Transcriptomic Analysis
RNA was extracted from resected livers, processed and analyzed as previously
described.17 Gene Ontology (GO) terms were analyzed using DAVID (NIAID).
Results
Estrogen enhances liver growth
To demonstrate the potential impact of endogenous estrogen levels on liver size, adult
zebrafish livers were assessed in females and males. Livers of adult female zebrafish
were bigger than that of male siblings, as visualized in hepatocyte reporter fish
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expressing GFP regulated by the fatty acid binding protein 10a promoter,
Tg(fabp10a:GFP) (Figure 1A). Baseline liver:body weight ratios in 10 WT males and
females at 7 months, revealed a significant 2.6-fold difference (Figure 1B). Since
estrogen is one of the major sex hormones determining physiological differences between
males and femails, the potential impact of estrogen on liver growth was examined:
zebrafish were exposed to E2 (10µM) or DMSO daily for six weeks, and liver size and
weight were assessed in the context of total body length and mass (Figure 1A,B). Male
livers responded more significantly to E2 with a 4.8-fold increase in liver:body weight
ratio, while females exhibited a 1.6-fold enhancement (Figure 1B). These results suggest
that liver mass is directly responsive to estrogen.
To uncover the signals by which E2 affects liver size, liver transcriptome analysis was
performed with and without E2. Gene expression in male and female livers was different,
with a subset of genes identified as “female-associated”, indicating genes up-regulated
exclusively in females (Supplementary Figure S1A). These differences, however, are
not solely due to estrogenic effects, as E2 did not simply convert the male liver
transcriptome into one resembling a female: in fact, E2 exposure altered transcriptional
profiles in both sexes (Supplementary Figure S1A). GO analysis revealed that E2
enhanced cell cycle and DNA metabolism-related gene expression (46 genes, p=3.4x10-
2%), TSC1/TSC2 (≤ 5%), and PTEN (≤ 3%).51, 52 In the context of our data, is tempting to
speculate that PI3K-mTOR activity in HCC may instead depend upon upstream ligand
activation, mediated by increased E2 levels and GPER1 expression in cirrhotics, as also
observewd by Wei et al. 41 As >80% of HCC patients are diagnosed at late stages without
hope for cure, there is an urgent need for earlier detection and targeted therapeutic
interventions. The pro-proliferative consequences of E2-GPER1 activation of PI3K-mTOR
signaling, together with our in vivo data showing strong responses to GPER1 antagonist
treatment in both cancer initiation and progression, clearly indicate that drugs targeting
E2-GPER1 may offer exciting new therapeutic applications in liver cancer prevention and
treatment.
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Figure Legends
Figure 1. E2 increases liver size
(A) Brightfield and fluorescent images of male and female Tg(fabp10a:GFP) adult
zebrafish exposed to DMSO (0.1%) or E2 (10µM) daily for 6 weeks. Scale bars, 2mm. (B)
Scheme illustrating DMBA carcinogenesis followed by chemical prevention trial. (G)
Kaplan–Meier survival plot. n = 52, 66, 39 for DMSO, E2, and E2+G-15 treated adults at
249 days post first treatment, p<0.0001, Log-rank (Mantel-Cox) test. (H) Liver tumor size
(mm) per fish at 9 mpf. *p=0.0172, **p=0.0053, ****p<0.0001, ns=not significant, one-way
ANOVA, stratified by gender.
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Summary: E2 signals via GPER1 to increase embryonic and adult liver size and growth by activation of PI3K signaling to mTOR. E2 signaling via GPER1 contributes to sex-dimorphic adult hepatocyte proliferation and promotes liver cancer formation and progression What you need to know Background: We investigated the effects of estrogen on hepatocyte proliferation during zebrafish development, liver regeneration, and carcinogenesis. We also studied human hepatocytes and liver tissues. Findings: Exposure to 17 beta-estradiol (E2) increased proliferation of hepatocytes and liver volume and mass in larval and adult zebrafish. GPER1 is a hepatic estrogen sensor that regulates liver growth during development, regeneration, and tumorigenesis. HCC samples from patients had increased levels of GPER1, compared with non-tumor tissues Limitations: This study was performed in an animal model of HCC. Implications for patients: Inhibitors of GPER1 might be developed for liver cancer prevention or treatment.
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Supporting Documents
Supplementary Figure Legends
Supplementary Figure S1. Estrogen increases liver growth and size.
(A) Venn diagram showing intersection of genes differentially expressed in the livers of male
vs. male+E2, female vs. female+E2, and female vs. male. (fold change >10). Values
demonstrate number of genes upregulated by E2 in male liver (299 genes), female liver (125
streptomycin (10,000 U/mL), at 37°C and 5% CO2. Fol lowing 24 h of serum starvation,
HepG2 cells were incubated with DMSO or G15 (500 nM) for 20 min prior to exposure to E2
(10nM), or E2+G15 for 30 min
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Supplementary Statistical Analysis
2-Way ANOVA analysis was performed where appropriate. Resulting p values for interaction
effects for each figure are listed below:
Figure 2F:
Two-way ANOVA Ordinary Alpha 0.05 Source of Variation % of total variation P value P value summary Significant? Interaction 1.017 <0.0001 **** Yes GPER1 Factor 88.56 <0.0001 **** Yes Age Factor 2.437 <0.0001 **** Yes ANOVA table SS (Type III) DF MS F (DFn, DFd) P value Interaction 1.202 2 0.6008 F (2, 414) = 46.48 P<0.0001 GPER1 Factor 104.6 2 52.31 F (2, 414) = 4046 P<0.0001 Age Factor 2.878 1 2.878 F (1, 414) = 222.7 P<0.0001 Residual 5.352 414 0.01293
Tukey's multiple comparisons test Mean Diff. 95.00% CI of diff. Significant? Summary Adjusted P Value
3dpf:gper1 +/+ vs. 3dpf:gper1 -/- 0.02061 -0.03554 to 0.07677 No ns 0.9002
3dpf:gper1 +/+ vs. 4dpf:gper1 +/+ -0.6682 -0.7344 to -0.6019 Yes **** <0.0001
3dpf:gper1 +/+ vs. 4dpf:gper1 -/- -0.4395 -0.4947 to -0.3842 Yes **** <0.0001
3dpf:gper1 +/+ vs. 5dpf:gper1 +/+ -1.446 -1.514 to -1.378 Yes **** <0.0001
3dpf:gper1 +/+ vs. 5dpf:gper1 -/- -1.164 -1.222 to -1.107 Yes **** <0.0001
3dpf:gper1 -/- vs. 4dpf:gper1 +/+ -0.6888 -0.7477 to -0.6299 Yes **** <0.0001
3dpf:gper1 -/- vs. 4dpf:gper1 -/- -0.4601 -0.5063 to -0.4139 Yes **** <0.0001
3dpf:gper1 -/- vs. 5dpf:gper1 +/+ -1.467 -1.527 to -1.406 Yes **** <0.0001
3dpf:gper1 -/- vs. 5dpf:gper1 -/- -1.185 -1.234 to -1.136 Yes **** <0.0001
4dpf:gper1 +/+ vs. 4dpf:gper1 -/- 0.2287 0.1706 to 0.2868 Yes **** <0.0001
4dpf:gper1 +/+ vs. 5dpf:gper1 +/+ -0.7779 -0.8481 to -0.7076 Yes **** <0.0001
4dpf:gper1 +/+ vs. 5dpf:gper1 -/- -0.496 -0.5562 to -0.4357 Yes **** <0.0001
4dpf:gper1 -/- vs. 5dpf:gper1 +/+ -1.007 -1.067 to -0.9465 Yes **** <0.0001
4dpf:gper1 -/- vs. 5dpf:gper1 -/- -0.7247 -0.7726 to -0.6768 Yes **** <0.0001
5dpf:gper1 +/+ vs. 5dpf:gper1 -/- 0.2819 0.2198 to 0.344 Yes **** <0.0001
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Figure 2H:
Two-way ANOVA Ordinary Alpha 0.05 Source of Variation % of total variation P value P value summary Significant? Interaction 4.535 0.0002 *** Yes GPER1 Factor 13.09 <0.0001 **** Yes Treatments Factor 32.08 <0.0001 **** Yes ANOVA table SS (Type III) DF MS F (DFn, DFd) P value Interaction 0.52 2 0.26 F (2, 182) = 8.847 P=0.0002 GPER1 Factor 1.502 2 0.7508 F (2, 182) = 25.55 P<0.0001 Treatments Factor 3.679 1 3.679 F (1, 182) = 125.2 P<0.0001 Residual 5.349 182 0.02939
Tukey's multiple comparisons test Mean Diff. 95.00% CI of diff. Significant? Summary Adjusted P Value
DMSO:gper1 +/+ vs. DMSO:gper1 -/- 0.1611 0.05204 to 0.2702 Yes *** 0.0005
DMSO:gper1 +/+ vs. E2:gper1 +/+ -0.3569 -0.4816 to -0.2322 Yes **** <0.0001
DMSO:gper1 +/+ vs. E2:gper1 -/- 0.07512 -0.08651 to 0.2367 No ns 0.7631
DMSO:gper1 +/+ vs. G-1:gper1 +/+ -0.2417 -0.3635 to -0.1199 Yes **** <0.0001
DMSO:gper1 +/+ vs. G-1:gper1 -/- 0.09102 -0.01674 to 0.1988 No ns 0.1505
DMSO:gper1 -/- vs. E2:gper1 +/+ -0.518 -0.6439 to -0.3921 Yes **** <0.0001
DMSO:gper1 -/- vs. E2:gper1 -/- -0.08602 -0.2485 to 0.0765 No ns 0.649
DMSO:gper1 -/- vs. G-1:gper1 +/+ -0.4028 -0.5258 to -0.2799 Yes **** <0.0001
DMSO:gper1 -/- vs. G-1:gper1 -/- -0.07012 -0.1792 to 0.03897 No ns 0.4357
E2:gper1 +/+ vs. E2:gper1 -/- 0.432 0.2586 to 0.6054 Yes **** <0.0001
E2:gper1 +/+ vs. G-1:gper1 +/+ 0.1152 -0.02188 to 0.2522 No ns 0.1547
E2:gper1 +/+ vs. G-1:gper1 -/- 0.4479 0.3232 to 0.5726 Yes **** <0.0001
E2:gper1 -/- vs. G-1:gper1 +/+ -0.3168 -0.4881 to -0.1455 Yes **** <0.0001
E2:gper1 -/- vs. G-1:gper1 -/- 0.0159 -0.1457 to 0.1775 No ns 0.9998
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Figure 4B:
Two-way ANOVA Ordinary Alpha 0.05 Source of Variation % of total variation P value P value summary Significant? Interaction 8.979 <0.0001 **** Yes GPER1 Factor 21.14 <0.0001 **** Yes Treatments Factor 28.76 <0.0001 **** Yes ANOVA table SS (Type III) DF MS F (DFn, DFd) P value Interaction 2.284 5 0.4567 F (5, 201) = 10.44 P<0.0001 GPER1 Factor 5.377 5 1.075 F (5, 201) = 24.58 P<0.0001 Treatments Factor 7.315 1 7.315 F (1, 201) = 167.2 P<0.0001 Residual 8.793 201 0.04375
.
Tukey's multiple comparisons test Mean Diff. 95.00% CI of diff. Significant? Summary Adjusted P Value
DMSO:gper1 +/+ vs. DMSO:gper1 -/- 0.1484 -0.04979 to 0.3466 No ns 0.361
DMSO:gper1 +/+ vs. G-1:gper1 +/+ -0.5409 -0.7451 to -0.3368 Yes **** <0.0001
DMSO:gper1 +/+ vs. G-1:gper1 -/- 0.07811 -0.1107 to 0.2669 No ns 0.9682
DMSO:gper1 +/+ vs. E2:gper1 +/+ -0.4441 -0.6815 to -0.2067 Yes **** <0.0001
DMSO:gper1 +/+ vs. E2:gper1 -/- 0.2086 -0.04509 to 0.4622 No ns 0.2247
DMSO:gper1 +/+ vs. MK:gper1 +/+ 0.1573 -0.06273 to 0.3772 No ns 0.4355
DMSO:gper1 +/+ vs. MK:gper1 -/- 0.2649 0.03977 to 0.49 Yes ** 0.0074
DMSO:gper1 +/+ vs. MK+E2:gper1 +/+ -0.1945 -0.4196 to 0.03061 No ns 0.1648
DMSO:gper1 +/+ vs. MK+E2:gper1 -/- 0.2754 0.08223 to 0.4685 Yes *** 0.0003
DMSO:gper1 +/+ vs. 740YP:gper1 +/+ -0.4384 -0.6315 to -0.2452 Yes **** <0.0001
DMSO:gper1 +/+ vs. 740YP:gper1 -/- -0.04803 -0.3242 to 0.2282 No ns >0.9999
DMSO:gper1 -/- vs. G-1:gper1 +/+ -0.6893 -0.9083 to -0.4703 Yes **** <0.0001
DMSO:gper1 -/- vs. G-1:gper1 -/- -0.07029 -0.275 to 0.1344 No ns 0.9927
DMSO:gper1 -/- vs. E2:gper1 +/+ -0.5925 -0.8428 to -0.3422 Yes **** <0.0001
DMSO:gper1 -/- vs. E2:gper1 -/- 0.06016 -0.2056 to 0.3259 No ns 0.9998
DMSO:gper1 -/- vs. MK:gper1 +/+ 0.008849 -0.225 to 0.2427 No ns >0.9999
DMSO:gper1 -/- vs. MK:gper1 -/- 0.1165 -0.1222 to 0.3551 No ns 0.9022
DMSO:gper1 -/- vs. MK+E2:gper1 +/+ -0.3429 -0.5815 to -0.1042 Yes *** 0.0002
DMSO:gper1 -/- vs. MK+E2:gper1 -/- 0.127 -0.08181 to 0.3357 No ns 0.6854
DMSO:gper1 -/- vs. 740YP:gper1 +/+ -0.5868 -0.7955 to -0.378 Yes **** <0.0001
DMSO:gper1 -/- vs. 740YP:gper1 -/- -0.1964 -0.4838 to 0.09095 No ns 0.5082
G-1:gper1 +/+ vs. G-1:gper1 -/- 0.6191 0.4085 to 0.8296 Yes **** <0.0001
G-1:gper1 +/+ vs. E2:gper1 +/+ 0.09681 -0.1582 to 0.3519 No ns 0.9835
G-1:gper1 +/+ vs. E2:gper1 -/- 0.7495 0.4793 to 1.02 Yes **** <0.0001
G-1:gper1 +/+ vs. MK:gper1 +/+ 0.6982 0.4593 to 0.9371 Yes **** <0.0001
G-1:gper1 +/+ vs. MK:gper1 -/- 0.8058 0.5622 to 1.049 Yes **** <0.0001
G-1:gper1 +/+ vs. MK+E2:gper1 +/+ 0.3464 0.1028 to 0.5901 Yes *** 0.0003
G-1:gper1 +/+ vs. MK+E2:gper1 -/- 0.8163 0.6019 to 1.031 Yes **** <0.0001
G-1:gper1 +/+ vs. 740YP:gper1 +/+ 0.1026 -0.1118 to 0.317 No ns 0.9137
G-1:gper1 +/+ vs. 740YP:gper1 -/- 0.4929 0.2014 to 0.7844 Yes **** <0.0001
G-1:gper1 -/- vs. E2:gper1 +/+ -0.5222 -0.7651 to -0.2793 Yes **** <0.0001
G-1:gper1 -/- vs. E2:gper1 -/- 0.1305 -0.1283 to 0.3893 No ns 0.8809
G-1:gper1 -/- vs. MK:gper1 +/+ 0.07914 -0.1468 to 0.305 No ns 0.9914
G-1:gper1 -/- vs. MK:gper1 -/- 0.1868 -0.04413 to 0.4177 No ns 0.2466
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G-1:gper1 -/- vs. MK+E2:gper1 +/+ -0.2726 -0.5035 to -0.04171 Yes ** 0.007
G-1:gper1 -/- vs. MK+E2:gper1 -/- 0.1973 -0.002591 to 0.3971 No ns 0.0567
G-1:gper1 -/- vs. 740YP:gper1 +/+ -0.5165 -0.7163 to -0.3166 Yes **** <0.0001
G-1:gper1 -/- vs. 740YP:gper1 -/- -0.1261 -0.4071 to 0.1548 No ns 0.9432
E2:gper1 +/+ vs. E2:gper1 -/- 0.6527 0.3565 to 0.9489 Yes **** <0.0001
E2:gper1 +/+ vs. MK:gper1 +/+ 0.6014 0.3335 to 0.8693 Yes **** <0.0001
E2:gper1 +/+ vs. MK:gper1 -/- 0.709 0.4369 to 0.9811 Yes **** <0.0001
E2:gper1 +/+ vs. MK+E2:gper1 +/+ 0.2496 -0.02247 to 0.5217 No ns 0.1066
E2:gper1 +/+ vs. MK+E2:gper1 -/- 0.7195 0.4732 to 0.9658 Yes **** <0.0001
E2:gper1 +/+ vs. 740YP:gper1 +/+ 0.005777 -0.2405 to 0.2521 No ns >0.9999
E2:gper1 +/+ vs. 740YP:gper1 -/- 0.3961 0.0804 to 0.7118 Yes ** 0.0028
E2:gper1 -/- vs. MK:gper1 +/+ -0.05131 -0.3337 to 0.2311 No ns >0.9999
E2:gper1 -/- vs. MK:gper1 -/- 0.05631 -0.2301 to 0.3427 No ns >0.9999
E2:gper1 -/- vs. MK+E2:gper1 +/+ -0.4031 -0.6894 to -0.1167 Yes *** 0.0004
E2:gper1 -/- vs. MK+E2:gper1 -/- 0.0668 -0.1952 to 0.3288 No ns 0.9995
E2:gper1 -/- vs. 740YP:gper1 +/+ -0.6469 -0.9089 to -0.3849 Yes **** <0.0001
E2:gper1 -/- vs. 740YP:gper1 -/- -0.2566 -0.5847 to 0.0715 No ns 0.2946
MK:gper1 +/+ vs. MK:gper1 -/- 0.1076 -0.1494 to 0.3647 No ns 0.9653
MK:gper1 +/+ vs. MK+E2:gper1 +/+ -0.3517 -0.6088 to -0.09471 Yes *** 0.0006
MK:gper1 +/+ vs. MK+E2:gper1 -/- 0.1181 -0.1114 to 0.3477 No ns 0.8656
MK:gper1 +/+ vs. 740YP:gper1 +/+ -0.5956 -0.8252 to -0.366 Yes **** <0.0001
MK:gper1 +/+ vs. 740YP:gper1 -/- -0.2053 -0.5081 to 0.09754 No ns 0.5216
MK:gper1 -/- vs. MK+E2:gper1 +/+ -0.4594 -0.7208 to -0.1979 Yes **** <0.0001
MK:gper1 -/- vs. MK+E2:gper1 -/- 0.01049 -0.224 to 0.245 No ns >0.9999
MK:gper1 -/- vs. 740YP:gper1 +/+ -0.7032 -0.9377 to -0.4688 Yes **** <0.0001
MK:gper1 -/- vs. 740YP:gper1 -/- -0.3129 -0.6195 to -0.006343 Yes * 0.0408
MK+E2:gper1 +/+ vs. MK+E2:gper1 -/- 0.4699 0.2354 to 0.7043 Yes **** <0.0001
MK+E2:gper1 +/+ vs. 740YP:gper1 +/+ -0.2439 -0.4783 to -0.009395 Yes * 0.0335
MK+E2:gper1 +/+ vs. 740YP:gper1 -/- 0.1465 -0.1601 to 0.453 No ns 0.9144
MK+E2:gper1 -/- vs. 740YP:gper1 +/+ -0.7137 -0.9177 to -0.5098 Yes **** <0.0001
MK+E2:gper1 -/- vs. 740YP:gper1 -/- -0.3234 -0.6073 to -0.03948 Yes * 0.0114
740YP:gper1 +/+ vs. 740YP:gper1 -/- 0.3903 0.1064 to 0.6742 Yes *** 0.0006
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Figure 4E:
Two-way ANOVA Ordinary Alpha 0.05
Source of Variation % of total variation P value
P value summary Significant?
Interaction 3.556 <0.0001 **** Yes GPER1 Factor 1.912 <0.0001 **** Yes Treatments Factor 76.97 <0.0001 **** Yes ANOVA table SS (Type III) DF MS F (DFn, DFd) P value Interaction 1.344 8 0.168 F (8, 196) = 5.922 P<0.0001 GPER1 Factor 0.7229 2 0.3615 F (2, 196) = 12.74 P<0.0001 Treatments Factor 29.1 4 7.275 F (4, 196) = 256.3 P<0.0001 Residual 5.562 196 0.02838
Tukey's multiple comparisons test
Mean Diff. 95.00% CI of diff. Significant?
Summary Adjusted P Value
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DMSO:+/+ vs. DMSO:-/- 0.1878 -0.04285 to 0.4184 No ns 0.2574 DMSO:+/+ vs. DMSO:+/+;+/- -0.05403 -0.2936 to 0.1856 No ns >0.9999 DMSO:+/+ vs. DMSO:+/+;-,- 0.6181 0.3652 to 0.8709 Yes **** <0.0001 DMSO:+/+ vs. DMSO:-/-;-/- 0.6099 0.3766 to 0.8432 Yes **** <0.0001 DMSO:+/+ vs. E2:+/+ -0.4318 -0.6847 to -0.179 Yes **** <0.0001 DMSO:+/+ vs. E2:-/- 0.1782 -0.07462 to 0.4311 No ns 0.5022 DMSO:+/+ vs. E2:+/+;+/- -0.3566 -0.5872 to -0.1259 Yes **** <0.0001 DMSO:+/+ vs. E2:+/+;-,- 0.6417 0.4055 to 0.878 Yes **** <0.0001 DMSO:+/+ vs. E2:-/-;-/- 0.6003 0.364 to 0.8365 Yes **** <0.0001 DMSO:+/+ vs. G-1:+/+ -0.4048 -0.6576 to -0.1519 Yes **** <0.0001 DMSO:+/+ vs. G-1:-/- 0.2116 -0.01663 to 0.4399 No ns 0.1018 DMSO:+/+ vs. G-1:+/+;+/- -0.1779 -0.3987 to 0.04276 No ns 0.2724 DMSO:+/+ vs. G-1:+/+;-,- 0.6256 0.3822 to 0.869 Yes **** <0.0001 DMSO:+/+ vs. G-1:-/-;-/- 0.6324 0.3736 to 0.8912 Yes **** <0.0001 DMSO:-/- vs. DMSO:+/+;+/- -0.2418 -0.4507 to -0.03295 Yes ** 0.0083 DMSO:-/- vs. DMSO:+/+;-,- 0.4303 0.2064 to 0.6542 Yes **** <0.0001 DMSO:-/- vs. DMSO:-/-;-/- 0.4221 0.2205 to 0.6237 Yes **** <0.0001 DMSO:-/- vs. E2:+/+ -0.6196 -0.8435 to -0.3956 Yes **** <0.0001 DMSO:-/- vs. E2:-/- -0.009545 -0.2335 to 0.2144 No ns >0.9999 DMSO:-/- vs. E2:+/+;+/- -0.5443 -0.7428 to -0.3458 Yes **** <0.0001 DMSO:-/- vs. E2:+/+;-,- 0.454 0.249 to 0.659 Yes **** <0.0001 DMSO:-/- vs. E2:-/-;-/- 0.4125 0.2075 to 0.6175 Yes **** <0.0001 DMSO:-/- vs. G-1:+/+ -0.5926 -0.8165 to -0.3686 Yes **** <0.0001 DMSO:-/- vs. G-1:-/- 0.02384 -0.1719 to 0.2196 No ns >0.9999 DMSO:-/- vs. G-1:+/+;+/- -0.3657 -0.5526 to -0.1788 Yes **** <0.0001 DMSO:-/- vs. G-1:+/+;-,- 0.4378 0.2246 to 0.651 Yes **** <0.0001 DMSO:-/- vs. G-1:-/-;-/- 0.4447 0.214 to 0.6753 Yes **** <0.0001 DMSO:+/+;+/- vs. DMSO:+/+;-,- 0.6721 0.4389 to 0.9053 Yes **** <0.0001 DMSO:+/+;+/- vs. DMSO:-/-;-/- 0.6639 0.4521 to 0.8757 Yes **** <0.0001 DMSO:+/+;+/- vs. E2:+/+ -0.3778 -0.6109 to -0.1446 Yes **** <0.0001 DMSO:+/+;+/- vs. E2:-/- 0.2323 -0.0009056 to 0.4654 No ns 0.0520 DMSO:+/+;+/- vs. E2:+/+;+/- -0.3025 -0.5114 to -0.09368 Yes *** 0.0001 DMSO:+/+;+/- vs. E2:+/+;-,- 0.6958 0.4807 to 0.9108 Yes **** <0.0001 DMSO:+/+;+/- vs. E2:-/-;-/- 0.6543 0.4392 to 0.8693 Yes **** <0.0001 DMSO:+/+;+/- vs. G-1:+/+ -0.3508 -0.5839 to -0.1176 Yes **** <0.0001 DMSO:+/+;+/- vs. G-1:-/- 0.2656 0.05942 to 0.4719 Yes ** 0.0015 DMSO:+/+;+/- vs. G-1:+/+;+/- -0.1239 -0.3218 to 0.07393 No ns 0.6984 DMSO:+/+;+/- vs. G-1:+/+;-,- 0.6796 0.4567 to 0.9025 Yes **** <0.0001 DMSO:+/+;+/- vs. G-1:-/-;-/- 0.6865 0.4469 to 0.9261 Yes **** <0.0001 DMSO:+/+;-,- vs. DMSO:-/-;-/- -0.008193 -0.2349 to 0.2185 No ns >0.9999 DMSO:+/+;-,- vs. E2:+/+ -1.05 -1.297 to -0.8031 Yes **** <0.0001 DMSO:+/+;-,- vs. E2:-/- -0.4398 -0.6866 to -0.1931 Yes **** <0.0001 DMSO:+/+;-,- vs. E2:+/+;+/- -0.9746 -1.199 to -0.7507 Yes **** <0.0001 DMSO:+/+;-,- vs. E2:+/+;-,- 0.02365 -0.2061 to 0.2534 No ns >0.9999 DMSO:+/+;-,- vs. E2:-/-;-/- -0.01781 -0.2475 to 0.2119 No ns >0.9999 DMSO:+/+;-,- vs. G-1:+/+ -1.023 -1.27 to -0.7761 Yes **** <0.0001 DMSO:+/+;-,- vs. G-1:-/- -0.4065 -0.6279 to -0.185 Yes **** <0.0001 DMSO:+/+;-,- vs. G-1:+/+;+/- -0.796 -1.01 to -0.5823 Yes **** <0.0001 DMSO:+/+;-,- vs. G-1:+/+;-,- 0.007514 -0.2296 to 0.2446 No ns >0.9999 DMSO:+/+;-,- vs. G-1:-/-;-/- 0.01437 -0.2385 to 0.2672 No ns >0.9999 DMSO:-/-;-/- vs. E2:+/+ -1.042 -1.268 to -0.815 Yes **** <0.0001 DMSO:-/-;-/- vs. E2:-/- -0.4317 -0.6583 to -0.205 Yes **** <0.0001 DMSO:-/-;-/- vs. E2:+/+;+/- -0.9664 -1.168 to -0.7649 Yes **** <0.0001 DMSO:-/-;-/- vs. E2:+/+;-,- 0.03185 -0.1761 to 0.2398 No ns >0.9999 DMSO:-/-;-/- vs. E2:-/-;-/- -0.009622 -0.2176 to 0.1984 No ns >0.9999 DMSO:-/-;-/- vs. G-1:+/+ -1.015 -1.241 to -0.788 Yes **** <0.0001
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Figure 5C:
Two-way ANOVA Ordinary Alpha 0.05 Source of Variation % of total variation P value P value summary Significant?
DMSO:-/-;-/- vs. G-1:-/- -0.3983 -0.5971 to -0.1994 Yes **** <0.0001 DMSO:-/-;-/- vs. G-1:+/+;+/- -0.7878 -0.978 to -0.5977 Yes **** <0.0001 DMSO:-/-;-/- vs. G-1:+/+;-,- 0.01571 -0.2004 to 0.2318 No ns >0.9999 DMSO:-/-;-/- vs. G-1:-/-;-/- 0.02256 -0.2107 to 0.2558 No ns >0.9999 E2:+/+ vs. E2:-/- 0.61 0.3633 to 0.8568 Yes **** <0.0001 E2:+/+ vs. E2:+/+;+/- 0.07524 -0.1487 to 0.2992 No ns 0.9980 E2:+/+ vs. E2:+/+;-,- 1.074 0.8438 to 1.303 Yes **** <0.0001 E2:+/+ vs. E2:-/-;-/- 1.032 0.8023 to 1.262 Yes **** <0.0001 E2:+/+ vs. G-1:+/+ 0.02702 -0.2197 to 0.2738 No ns >0.9999 E2:+/+ vs. G-1:-/- 0.6434 0.4219 to 0.8649 Yes **** <0.0001 E2:+/+ vs. G-1:+/+;+/- 0.2539 0.04016 to 0.4676 Yes ** 0.0056 E2:+/+ vs. G-1:+/+;-,- 1.057 0.8203 to 1.294 Yes **** <0.0001 E2:+/+ vs. G-1:-/-;-/- 1.064 0.8114 to 1.317 Yes **** <0.0001 E2:-/- vs. E2:+/+;+/- -0.5348 -0.7587 to -0.3109 Yes **** <0.0001 E2:-/- vs. E2:+/+;-,- 0.4635 0.2338 to 0.6932 Yes **** <0.0001 E2:-/- vs. E2:-/-;-/- 0.422 0.1923 to 0.6518 Yes **** <0.0001 E2:-/- vs. G-1:+/+ -0.583 -0.8298 to -0.3363 Yes **** <0.0001 E2:-/- vs. G-1:-/- 0.03338 -0.1881 to 0.2549 No ns >0.9999 E2:-/- vs. G-1:+/+;+/- -0.3562 -0.5699 to -0.1425 Yes **** <0.0001 E2:-/- vs. G-1:+/+;-,- 0.4474 0.2103 to 0.6844 Yes **** <0.0001 E2:-/- vs. G-1:-/-;-/- 0.4542 0.2014 to 0.7071 Yes **** <0.0001 E2:+/+;+/- vs. E2:+/+;-,- 0.9983 0.7933 to 1.203 Yes **** <0.0001 E2:+/+;+/- vs. E2:-/-;-/- 0.9568 0.7518 to 1.162 Yes **** <0.0001 E2:+/+;+/- vs. G-1:+/+ -0.04823 -0.2722 to 0.1757 No ns >0.9999 E2:+/+;+/- vs. G-1:-/- 0.5682 0.3725 to 0.7639 Yes **** <0.0001 E2:+/+;+/- vs. G-1:+/+;+/- 0.1786 -0.008261 to 0.3655 No ns 0.0778 E2:+/+;+/- vs. G-1:+/+;-,- 0.9822 0.7689 to 1.195 Yes **** <0.0001 E2:+/+;+/- vs. G-1:-/-;-/- 0.989 0.7584 to 1.22 Yes **** <0.0001 E2:+/+;-,- vs. E2:-/-;-/- -0.04147 -0.2528 to 0.1698 No ns >0.9999 E2:+/+;-,- vs. G-1:+/+ -1.047 -1.276 to -0.8168 Yes **** <0.0001 E2:+/+;-,- vs. G-1:-/- -0.4301 -0.6324 to -0.2278 Yes **** <0.0001 E2:+/+;-,- vs. G-1:+/+;+/- -0.8197 -1.013 to -0.6259 Yes **** <0.0001 E2:+/+;-,- vs. G-1:+/+;-,- -0.01614 -0.2354 to 0.2031 No ns >0.9999 E2:+/+;-,- vs. G-1:-/-;-/- -0.009285 -0.2455 to 0.227 No ns >0.9999 E2:-/-;-/- vs. G-1:+/+ -1.005 -1.235 to -0.7753 Yes **** <0.0001 E2:-/-;-/- vs. G-1:-/- -0.3887 -0.591 to -0.1863 Yes **** <0.0001 E2:-/-;-/- vs. G-1:+/+;+/- -0.7782 -0.972 to -0.5844 Yes **** <0.0001 E2:-/-;-/- vs. G-1:+/+;-,- 0.02533 -0.194 to 0.2446 No ns >0.9999 E2:-/-;-/- vs. G-1:-/-;-/- 0.03218 -0.2041 to 0.2684 No ns >0.9999 G-1:+/+ vs. G-1:-/- 0.6164 0.3949 to 0.8379 Yes **** <0.0001 G-1:+/+ vs. G-1:+/+;+/- 0.2268 0.01314 to 0.4405 Yes * 0.0258 G-1:+/+ vs. G-1:+/+;-,- 1.03 0.7933 to 1.267 Yes **** <0.0001 G-1:+/+ vs. G-1:-/-;-/- 1.037 0.7844 to 1.29 Yes **** <0.0001 G-1:-/- vs. G-1:+/+;+/- -0.3896 -0.5735 to -0.2056 Yes **** <0.0001 G-1:-/- vs. G-1:+/+;-,- 0.414 0.2033 to 0.6246 Yes **** <0.0001 G-1:-/- vs. G-1:-/-;-/- 0.4208 0.1926 to 0.6491 Yes **** <0.0001 G-1:+/+;+/- vs. G-1:+/+;-,- 0.8035 0.6011 to 1.006 Yes **** <0.0001 G-1:+/+;+/- vs. G-1:-/-;-/- 0.8104 0.5897 to 1.031 Yes **** <0.0001 G-1:+/+;-,- vs. G-1:-/-;-/- 0.006855 -0.2366 to 0.2503 No ns >0.9999
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Interaction 4.244 <0.0001 **** Yes Treatment Factor 1.905 0.0010 ** Yes Genetic Factor 69.46 <0.0001 **** Yes ANOVA table SS (Type III) DF MS F (DFn, DFd) P value Interaction 0.1113 6 0.01854 F (6, 185) = 5.303 P<0.0001 Treatment Factor 0.04994 2 0.02497 F (2, 185) = 7.141 P=0.0010 Genetic Factor 1.821 3 0.6069 F (3, 185) = 173.6 P<0.0001 Residual 0.6469 185 0.003497
Tukey's multiple comparisons test Mean Diff. 95.00% CI of diff. Significant? Summary Adjusted P Value
DMSO:gper1 +/+ ; mtor +/+ vs. DMSO:gper1 -/- ; mtor +/+ 0.09214 0.03008 to 0.1542 Yes *** 0.0001
DMSO:gper1 +/+ ; mtor +/+ vs. DMSO:gper1 +/+ ; mtor +/- 0.05953 -0.003083 to 0.1221 No ns 0.0793
DMSO:gper1 +/+ ; mtor +/+ vs. DMSO:gper1 -/- ; mtor -/- 0.2281 0.1543 to 0.3019 Yes **** <0.0001
DMSO:gper1 +/+ ; mtor +/+ vs. E2:gper1 +/+ ; mtor +/+ -0.08155 -0.1487 to -0.0144 Yes ** 0.0047
DMSO:gper1 +/+ ; mtor +/+ vs. E2:gper1 -/- ; mtor +/+ 0.1088 0.04677 to 0.1709 Yes **** <0.0001
DMSO:gper1 +/+ ; mtor +/+ vs. E2:gper1 +/+ ; mtor +/- -0.00843 -0.08418 to 0.06732 No ns >0.9999
DMSO:gper1 +/+ ; mtor +/+ vs. E2:gper1 -/- ; mtor -/- 0.23 0.146 to 0.3139 Yes **** <0.0001
DMSO:gper1 +/+ ; mtor +/+ vs. G-1:gper1 +/+ ; mtor +/+ -0.1223 -0.1961 to -0.04849 Yes **** <0.0001
DMSO:gper1 +/+ ; mtor +/+ vs. G-1:gper1 -/- ; mtor +/+ 0.08963 0.02642 to 0.1528 Yes *** 0.0003
DMSO:gper1 +/+ ; mtor +/+ vs. G-1:gper1 +/+ ; mtor +/- 0.03514 -0.03866 to 0.109 No ns 0.9155
DMSO:gper1 +/+ ; mtor +/+ vs. G-1:gper1 -/- ; mtor -/- 0.2356 0.1662 to 0.3049 Yes **** <0.0001
DMSO:gper1 -/- ; mtor +/+ vs. DMSO:gper1 +/+ ; mtor +/- -0.03261 -0.08973 to 0.02451 No ns 0.7641
DMSO:gper1 -/- ; mtor +/+ vs. DMSO:gper1 -/- ; mtor -/- 0.136 0.06677 to 0.2052 Yes **** <0.0001
DMSO:gper1 -/- ; mtor +/+ vs. E2:gper1 +/+ ; mtor +/+ -0.1737 -0.2357 to -0.1116 Yes **** <0.0001
DMSO:gper1 -/- ; mtor +/+ vs. E2:gper1 -/- ; mtor +/+ 0.01669 -0.03982 to 0.0732 No ns 0.998
DMSO:gper1 -/- ; mtor +/+ vs. E2:gper1 +/+ ; mtor +/- -0.1006 -0.1718 to -0.02929 Yes *** 0.0004
DMSO:gper1 -/- ; mtor +/+ vs. E2:gper1 -/- ; mtor -/- 0.1378 0.05791 to 0.2177 Yes **** <0.0001
DMSO:gper1 -/- ; mtor +/+ vs. G-1:gper1 +/+ ; mtor +/+ -0.2144 -0.2836 to -0.1452 Yes **** <0.0001
DMSO:gper1 -/- ; mtor +/+ vs. G-1:gper1 -/- ; mtor +/+ -0.002503 -0.06028 to 0.05528 No ns >0.9999
DMSO:gper1 -/- ; mtor +/+ vs. G-1:gper1 +/+ ; mtor +/- -0.05699 -0.1262 to 0.01222 No ns 0.2221
DMSO:gper1 -/- ; mtor +/+ vs. G-1:gper1 -/- ; mtor -/- 0.1435 0.07903 to 0.2079 Yes **** <0.0001
DMSO:gper1 +/+ ; mtor +/- vs. DMSO:gper1 -/- ; mtor -/- 0.1686 0.09888 to 0.2383 Yes **** <0.0001
DMSO:gper1 +/+ ; mtor +/- vs. E2:gper1 +/+ ; mtor +/+ -0.1411 -0.2037 to -0.07846 Yes **** <0.0001
DMSO:gper1 +/+ ; mtor +/- vs. E2:gper1 -/- ; mtor +/+ 0.0493 -0.007823 to 0.1064 No ns 0.1656
DMSO:gper1 +/+ ; mtor +/- vs. E2:gper1 +/+ ; mtor +/- -0.06796 -0.1397 to 0.003803 No ns 0.0821
DMSO:gper1 +/+ ; mtor +/- vs. E2:gper1 -/- ; mtor -/- 0.1704 0.09008 to 0.2508 Yes **** <0.0001
DMSO:gper1 +/+ ; mtor +/- vs. G-1:gper1 +/+ ; mtor +/+ -0.1818 -0.2515 to -0.1121 Yes **** <0.0001
DMSO:gper1 +/+ ; mtor +/- vs. G-1:gper1 -/- ; mtor +/+ 0.0301 -0.02827 to 0.08848 No ns 0.8629
DMSO:gper1 +/+ ; mtor +/- vs. G-1:gper1 +/+ ; mtor +/- -0.02438 -0.0941 to 0.04533 No ns 0.9913
DMSO:gper1 +/+ ; mtor +/- vs. G-1:gper1 -/- ; mtor -/- 0.1761 0.1111 to 0.241 Yes **** <0.0001
DMSO:gper1 -/- ; mtor -/- vs. E2:gper1 +/+ ; mtor +/+ -0.3097 -0.3835 to -0.2359 Yes **** <0.0001
DMSO:gper1 -/- ; mtor -/- vs. E2:gper1 -/- ; mtor +/+ -0.1193 -0.1885 to -0.05008 Yes **** <0.0001
DMSO:gper1 -/- ; mtor -/- vs. E2:gper1 +/+ ; mtor +/- -0.2365 -0.3183 to -0.1548 Yes **** <0.0001
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DMSO:gper1 -/- ; mtor -/- vs. E2:gper1 -/- ; mtor -/- 0.001846 -0.0875 to 0.0912 No ns >0.9999
DMSO:gper1 -/- ; mtor -/- vs. G-1:gper1 +/+ ; mtor +/+ -0.3504 -0.4303 to -0.2705 Yes **** <0.0001
DMSO:gper1 -/- ; mtor -/- vs. G-1:gper1 -/- ; mtor +/+ -0.1385 -0.2087 to -0.06823 Yes **** <0.0001
DMSO:gper1 -/- ; mtor -/- vs. G-1:gper1 +/+ ; mtor +/- -0.193 -0.2729 to -0.1131 Yes **** <0.0001
DMSO:gper1 -/- ; mtor -/- vs. G-1:gper1 -/- ; mtor -/- 0.007476 -0.06834 to 0.08329 No ns >0.9999
E2:gper1 +/+ ; mtor +/+ vs. E2:gper1 -/- ; mtor +/+ 0.1904 0.1283 to 0.2524 Yes **** <0.0001
E2:gper1 +/+ ; mtor +/+ vs. E2:gper1 +/+ ; mtor +/- 0.07312 -0.002633 to 0.1489 No ns 0.0695
E2:gper1 +/+ ; mtor +/+ vs. E2:gper1 -/- ; mtor -/- 0.3115 0.2276 to 0.3954 Yes **** <0.0001
E2:gper1 +/+ ; mtor +/+ vs. G-1:gper1 +/+ ; mtor +/+ -0.04075 -0.1146 to 0.03306 No ns 0.8007
E2:gper1 +/+ ; mtor +/+ vs. G-1:gper1 -/- ; mtor +/+ 0.1712 0.108 to 0.2344 Yes **** <0.0001
E2:gper1 +/+ ; mtor +/+ vs. G-1:gper1 +/+ ; mtor +/- 0.1167 0.04288 to 0.1905 Yes **** <0.0001
E2:gper1 +/+ ; mtor +/+ vs. G-1:gper1 -/- ; mtor -/- 0.3171 0.2478 to 0.3865 Yes **** <0.0001
E2:gper1 -/- ; mtor +/+ vs. E2:gper1 +/+ ; mtor +/- -0.1173 -0.1885 to -0.04598 Yes **** <0.0001
E2:gper1 -/- ; mtor +/+ vs. E2:gper1 -/- ; mtor -/- 0.1211 0.04122 to 0.2011 Yes **** <0.0001
E2:gper1 -/- ; mtor +/+ vs. G-1:gper1 +/+ ; mtor +/+ -0.2311 -0.3003 to -0.1619 Yes **** <0.0001
E2:gper1 -/- ; mtor +/+ vs. G-1:gper1 -/- ; mtor +/+ -0.01919 -0.07697 to 0.03859 No ns 0.9944
E2:gper1 -/- ; mtor +/+ vs. G-1:gper1 +/+ ; mtor +/- -0.07368 -0.1429 to -0.004472 Yes * 0.026
E2:gper1 -/- ; mtor +/+ vs. G-1:gper1 -/- ; mtor -/- 0.1268 0.06234 to 0.1912 Yes **** <0.0001
E2:gper1 +/+ ; mtor +/- vs. E2:gper1 -/- ; mtor -/- 0.2384 0.1474 to 0.3294 Yes **** <0.0001
E2:gper1 +/+ ; mtor +/- vs. G-1:gper1 +/+ ; mtor +/+ -0.1139 -0.1956 to -0.03215 Yes *** 0.0004
E2:gper1 +/+ ; mtor +/- vs. G-1:gper1 -/- ; mtor +/+ 0.09806 0.02578 to 0.1704 Yes *** 0.0007
E2:gper1 +/+ ; mtor +/- vs. G-1:gper1 +/+ ; mtor +/- 0.04357 -0.03814 to 0.1253 No ns 0.8344
E2:gper1 +/+ ; mtor +/- vs. G-1:gper1 -/- ; mtor -/- 0.244 0.1663 to 0.3217 Yes **** <0.0001
E2:gper1 -/- ; mtor -/- vs. G-1:gper1 +/+ ; mtor +/+ -0.3523 -0.4416 to -0.2629 Yes **** <0.0001
E2:gper1 -/- ; mtor -/- vs. G-1:gper1 -/- ; mtor +/+ -0.1403 -0.2212 to -0.05951 Yes **** <0.0001
E2:gper1 -/- ; mtor -/- vs. G-1:gper1 +/+ ; mtor +/- -0.1948 -0.2842 to -0.1055 Yes **** <0.0001
E2:gper1 -/- ; mtor -/- vs. G-1:gper1 -/- ; mtor -/- 0.00563 -0.08007 to 0.09133 No ns >0.9999
G-1:gper1 +/+ ; mtor +/+ vs. G-1:gper1 -/- ; mtor +/+ 0.2119 0.1417 to 0.2822 Yes **** <0.0001
G-1:gper1 +/+ ; mtor +/+ vs. G-1:gper1 +/+ ; mtor +/- 0.1574 0.07752 to 0.2374 Yes **** <0.0001
G-1:gper1 +/+ ; mtor +/+ vs. G-1:gper1 -/- ; mtor -/- 0.3579 0.2821 to 0.4337 Yes **** <0.0001
G-1:gper1 -/- ; mtor +/+ vs. G-1:gper1 +/+ ; mtor +/- -0.05449 -0.1247 to 0.01576 No ns 0.3059
G-1:gper1 -/- ; mtor +/+ vs. G-1:gper1 -/- ; mtor -/- 0.146 0.08041 to 0.2115 Yes **** <0.0001
G-1:gper1 +/+ ; mtor +/- vs. G-1:gper1 -/- ; mtor -/- 0.2005 0.1246 to 0.2763 Yes **** <0.0001
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Figure 5E:
Two-way ANOVA Ordinary Alpha 0.05 Source of Variation % of total variation P value P value summary Significant? Interaction 1.613 0.2150 ns No Mtz Factor 0.01651 0.8581 ns No Genetic Factor 58.51 <0.0001 **** Yes ANOVA table SS (Type III) DF MS F (DFn, DFd) P value Interaction 1122 2 561.1 F (2, 68) = 1.572 P=0.2150 Mtz Factor 11.49 1 11.49 F (1, 68) = 0.03219 P=0.8581 Genetic Factor 40711 2 20356 F (2, 68) = 57.04 P<0.0001 Residual 24269 68 356.9
Sidak's multiple comparisons test Mean Diff. 95.00% CI of diff. Significant? Summary Adjusted P Value
No MTz:gper1 +/+ vs. No MTz:gper1 -/- 12.37 -11.26 to 36 No ns 0.845
No MTz:gper1 +/+ vs. No MTz:ztor MUT 50.54 28.92 to 72.15 Yes **** <0.0001
No MTz:gper1 +/+ vs. MTz:gper1 +/+ -11.11 -29.44 to 7.229 No ns 0.6657
No MTz:gper1 +/+ vs. MTz:gper1 -/- 13.27 -12.47 to 39.01 No ns 0.8589
No MTz:gper1 +/+ vs. MTz:ztor MUT 58.1 30.88 to 85.32 Yes **** <0.0001
No MTz:gper1 -/- vs. No MTz:ztor MUT 38.17 12.89 to 63.44 Yes *** 0.0003
No MTz:gper1 -/- vs. MTz:gper1 +/+ -23.47 -46.01 to -0.9364 Yes * 0.0347
No MTz:gper1 -/- vs. MTz:gper1 -/- 0.9026 -27.99 to 29.79 No ns >0.9999
No MTz:gper1 -/- vs. MTz:ztor MUT 45.73 15.52 to 75.94 Yes *** 0.0003
No MTz:ztor MUT vs. MTz:gper1 +/+ -61.64 -82.06 to -41.23 Yes **** <0.0001
No MTz:ztor MUT vs. MTz:gper1 -/- -37.27 -64.53 to -10 Yes ** 0.0014No MTz:ztor MUT vs. MTz:ztor MUT 7.564 -21.1 to 36.22 No ns 0.9998
MTz:gper1 +/+ vs. MTz:gper1 -/- 24.38 -0.3672 to 49.12 No ns 0.0568
MTz:gper1 +/+ vs. MTz:ztor MUT 69.21 42.93 to 95.48 Yes **** <0.0001
MTz:gper1 -/- vs. MTz:ztor MUT 44.83 12.94 to 76.72 Yes *** 0.0009
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Figure 6B:
Male
Two-way ANOVA Ordinary Alpha 0.05 Source of Variation % of total variation P value P value summary Significant? Interaction 8.354 0.0005 *** Yes GPER1 Factor 68.38 <0.0001 **** Yes E2 Treatment Factor 6.569 0.0018 ** Yes ANOVA table SS (Type III) DF MS F (DFn, DFd) P value Interaction 5.025 1 5.025 F (1, 36) = 14.4 P=0.0005 GPER1 Factor 41.13 1 41.13 F (1, 36) = 117.9 P<0.0001 E2 Treatment Factor 3.951 1 3.951 F (1, 36) = 11.32 P=0.0018 Residual 12.56 36 0.3489
Tukey's multiple comparisons test Mean Diff. 95.00% CI of diff. Significant? Summary Adjusted P Value
DMSO:gper1 +/+ vs. DMSO:gper1 -/- -0.08048 -0.7919 to 0.631 No ns 0.99
DMSO:gper1 +/+ vs. E2:gper1 +/+ -2.744 -3.475 to -2.013 Yes **** <0.0001
DMSO:gper1 +/+ vs. E2:gper1 -/- -1.403 -2.098 to -0.7078 Yes **** <0.0001
DMSO:gper1 -/- vs. E2:gper1 +/+ -2.663 -3.394 to -1.932 Yes **** <0.0001
DMSO:gper1 -/- vs. E2:gper1 -/- -1.322 -2.018 to -0.6274 Yes **** <0.0001
E2:gper1 +/+ vs. E2:gper1 -/- 1.341 0.6258 to 2.056 Yes **** <0.0001
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Figure 6B:
Female
Two-way ANOVA Ordinary Alpha 0.05 Source of Variation % of total variation P value P value summary Significant? Interaction 0.1474 0.7367 ns No GPER1 Factor 56.59 <0.0001 **** Yes E2 Treatment Factor 5.063 0.0566 ns No ANOVA table SS (Type III) DF MS F (DFn, DFd) P value Interaction 0.03853 1 0.03853 F (1, 27) = 0.1154 P=0.7367 GPER1 Factor 14.79 1 14.79 F (1, 27) = 44.33 P<0.0001 E2 Treatment Factor 1.324 1 1.324 F (1, 27) = 3.966 P=0.0566 Residual 9.011 27 0.3337
Tukey's multiple comparisons test Mean Diff. 95.00% CI of diff. Significant? Summary Adjusted P Value
DMSO:gper1 +/+ vs. DMSO:gper1 -/- 0.4994 -0.2505 to 1.249 No ns 0.2851
DMSO:gper1 +/+ vs. E2:gper1 +/+ -1.353 -2.103 to -0.6036 Yes *** 0.0002
DMSO:gper1 +/+ vs. E2:gper1 -/- -0.9997 -1.866 to -0.1338 Yes * 0.0191
DMSO:gper1 -/- vs. E2:gper1 +/+ -1.853 -2.643 to -1.062 Yes **** <0.0001
DMSO:gper1 -/- vs. E2:gper1 -/- -1.499 -2.4 to -0.5978 Yes *** 0.0006
E2:gper1 +/+ vs. E2:gper1 -/- 0.3538 -0.5474 to 1.255 No ns 0.7077
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Figure 7H:
One-way ANOVA
Male Number of families 1 Number of comparisons per family 6 Alpha 0.05 Tukey's multiple comparisons test Mean Diff. 95.00% CI of diff. Significant? Summary Adjusted P Value DMSO vs. E2 -0.7606 -1.42 to -0.1013 Yes * 0.0172 DMSO vs. E2 +G15 -0.08686 -0.7634 to 0.5897 No ns 0.9867 DMSO vs. E2 +Ful -2.092 -3.086 to -1.097 Yes **** <0.0001 E2 vs. E2 +G15 0.6737 -0.03891 to 1.386 No ns 0.0707 E2 vs. E2 +Ful -1.331 -2.35 to -0.3119 Yes ** 0.0053 E2 +G15 vs. E2 +Ful -2.005 -3.035 to -0.9743 Yes **** <0.0001 Female
Number of families 1 Number of comparisons per family 6 Alpha 0.05 Tukey's multiple comparisons test Mean Diff. 95.00% CI of diff. Significant? Summary Adjusted P Value DMSO vs. E2 -0.045 -1.011 to 0.9207 No ns 0.9993 DMSO vs. E2 +G15 0.3333 -0.9571 to 1.624 No ns 0.9032 DMSO vs. E2 +Ful -0.05705 -1.189 to 1.075 No ns 0.9991 E2 vs. E2 +G15 0.3783 -0.7466 to 1.503 No ns 0.8105 E2 vs. E2 +Ful -0.01205 -0.9508 to 0.9267 No ns >0.9999 E2 +G15 vs. E2 +Ful -0.3904 -1.661 to 0.88 No ns 0.8484
Figure S2C:
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Two-way ANOVA Ordinary Alpha 0.05 Source of Variation % of total variation P value P value summary Significant? Interaction 5.219 <0.0001 **** Yes Treatment E2 Factor 34.16 <0.0001 **** Yes MOs Factor 20.17 <0.0001 **** Yes ANOVA table SS (Type III) DF MS F (DFn, DFd) P value Interaction 1.312 5 0.2624 F (5, 240) = 6.302 P<0.0001 Treatment E2 Factor 8.585 1 8.585 F (1, 240) = 206.2 P<0.0001 MOs Factor 5.07 5 1.014 F (5, 240) = 24.36 P<0.0001 Residual 9.991 240 0.04163
Tukey's multiple comparisons test Mean Diff. 95.00% CI of diff. Significant? Summary Adjusted P ValueDMSO:con vs. DMSO:esr1 -0.03659 -0.2231 to 0.1499 No ns >0.9999DMSO:con vs. DMSO:esr2a 0.1031 -0.08346 to 0.2896 No ns 0.8034DMSO:con vs. DMSO:esr2b 0.1161 -0.073 to 0.3053 No ns 0.6748DMSO:con vs. DMSO:esr2a+2b -0.04011 -0.2681 to 0.1879 No ns >0.9999DMSO:con vs. DMSO:gper1 0.1945 -0.003962 to 0.3929 No ns 0.0606DMSO:con vs. E2:con -0.4567 -0.6264 to -0.2869 Yes **** <0.0001DMSO:con vs. E2:esr1 -0.3783 -0.5845 to -0.1721 Yes **** <0.0001DMSO:con vs. E2:esr2a -0.3783 -0.5845 to -0.1721 Yes **** <0.0001DMSO:con vs. E2:esr2b -0.3375 -0.5359 to -0.1391 Yes **** <0.0001DMSO:con vs. E2:esr2a+2b -0.5442 -0.7657 to -0.3227 Yes **** <0.0001DMSO:con vs. E2:gper1 0.1051 -0.06462 to 0.2748 No ns 0.6628DMSO:esr1 vs. DMSO:esr2a 0.1396 -0.06341 to 0.3427 No ns 0.5009DMSO:esr1 vs. DMSO:esr2b 0.1527 -0.05274 to 0.3582 No ns 0.3748DMSO:esr1 vs. DMSO:esr2a+2b -0.003522 -0.2452 to 0.2382 No ns >0.9999DMSO:esr1 vs. DMSO:gper1 0.231 0.01701 to 0.4451 Yes * 0.022DMSO:esr1 vs. E2:con -0.4201 -0.6078 to -0.2323 Yes **** <0.0001DMSO:esr1 vs. E2:esr1 -0.3417 -0.563 to -0.1205 Yes **** <0.0001DMSO:esr1 vs. E2:esr2a -0.3417 -0.563 to -0.1205 Yes **** <0.0001DMSO:esr1 vs. E2:esr2b -0.3009 -0.5149 to -0.08686 Yes *** 0.0004DMSO:esr1 vs. E2:esr2a+2b -0.5076 -0.7432 to -0.272 Yes **** <0.0001DMSO:esr1 vs. E2:gper1 0.1417 -0.04605 to 0.3294 No ns 0.3507DMSO:esr2a vs. DMSO:esr2b 0.01308 -0.1924 to 0.2185 No ns >0.9999DMSO:esr2a vs. DMSO:esr2a+2b -0.1432 -0.3849 to 0.09852 No ns 0.7229DMSO:esr2a vs. DMSO:gper1 0.0914 -0.1226 to 0.3054 No ns 0.9608DMSO:esr2a vs. E2:con -0.5597 -0.7474 to -0.372 Yes **** <0.0001DMSO:esr2a vs. E2:esr1 -0.4814 -0.7027 to -0.2601 Yes **** <0.0001DMSO:esr2a vs. E2:esr2a -0.4814 -0.7027 to -0.2601 Yes **** <0.0001DMSO:esr2a vs. E2:esr2b -0.4405 -0.6546 to -0.2265 Yes **** <0.0001DMSO:esr2a vs. E2:esr2a+2b -0.6473 -0.8829 to -0.4117 Yes **** <0.0001DMSO:esr2a vs. E2:gper1 0.002041 -0.1857 to 0.1898 No ns >0.9999DMSO:esr2b vs. DMSO:esr2a+2b -0.1562 -0.4 to 0.08746 No ns 0.6118DMSO:esr2b vs. DMSO:gper1 0.07832 -0.138 to 0.2946 No ns 0.9889
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DMSO:esr2b vs. E2:con -0.5728 -0.7631 to -0.3824 Yes **** <0.0001DMSO:esr2b vs. E2:esr1 -0.4945 -0.7179 to -0.271 Yes **** <0.0001DMSO:esr2b vs. E2:esr2a -0.4945 -0.7179 to -0.271 Yes **** <0.0001DMSO:esr2b vs. E2:esr2b -0.4536 -0.6699 to -0.2373 Yes **** <0.0001DMSO:esr2b vs. E2:esr2a+2b -0.6603 -0.898 to -0.4227 Yes **** <0.0001DMSO:esr2b vs. E2:gper1 -0.01104 -0.2014 to 0.1793 No ns >0.9999DMSO:esr2a+2b vs. DMSO:gper1 0.2346 -0.01642 to 0.4856 No ns 0.092DMSO:esr2a+2b vs. E2:con -0.4165 -0.6455 to -0.1876 Yes **** <0.0001DMSO:esr2a+2b vs. E2:esr1 -0.3382 -0.5954 to -0.08104 Yes ** 0.0012DMSO:esr2a+2b vs. E2:esr2a -0.3382 -0.5954 to -0.08104 Yes ** 0.0012DMSO:esr2a+2b vs. E2:esr2b -0.2974 -0.5484 to -0.04639 Yes ** 0.0066DMSO:esr2a+2b vs. E2:esr2a+2b -0.5041 -0.7737 to -0.2345 Yes **** <0.0001DMSO:esr2a+2b vs. E2:gper1 0.1452 -0.08376 to 0.3742 No ns 0.6281DMSO:gper1 vs. E2:con -0.6511 -0.8507 to -0.4515 Yes **** <0.0001DMSO:gper1 vs. E2:esr1 -0.5728 -0.8042 to -0.3414 Yes **** <0.0001DMSO:gper1 vs. E2:esr2a -0.5728 -0.8042 to -0.3414 Yes **** <0.0001DMSO:gper1 vs. E2:esr2b -0.5319 -0.7564 to -0.3075 Yes **** <0.0001DMSO:gper1 vs. E2:esr2a+2b -0.7387 -0.9838 to -0.4935 Yes **** <0.0001DMSO:gper1 vs. E2:gper1 -0.08936 -0.2889 to 0.1102 No ns 0.9453E2:con vs. E2:esr1 0.07832 -0.129 to 0.2856 No ns 0.9844E2:con vs. E2:esr2a 0.07832 -0.129 to 0.2856 No ns 0.9844E2:con vs. E2:esr2b 0.1192 -0.0804 to 0.3187 No ns 0.7126E2:con vs. E2:esr2a+2b -0.08756 -0.3101 to 0.135 No ns 0.9786E2:con vs. E2:gper1 0.5617 0.3907 to 0.7328 Yes **** <0.0001E2:esr1 vs. E2:esr2a 0 -0.2381 to 0.2381 No ns >0.9999E2:esr1 vs. E2:esr2b 0.04084 -0.1906 to 0.2722 No ns >0.9999E2:esr1 vs. E2:esr2a+2b -0.1659 -0.4173 to 0.08558 No ns 0.5677E2:esr1 vs. E2:gper1 0.4834 0.2761 to 0.6907 Yes **** <0.0001E2:esr2a vs. E2:esr2b 0.04084 -0.1906 to 0.2722 No ns >0.9999E2:esr2a vs. E2:esr2a+2b -0.1659 -0.4173 to 0.08558 No ns 0.5677E2:esr2a vs. E2:gper1 0.4834 0.2761 to 0.6907 Yes **** <0.0001E2:esr2b vs. E2:esr2a+2b -0.2067 -0.4518 to 0.0384 No ns 0.1938E2:esr2b vs. E2:gper1 0.4426 0.243 to 0.6422 Yes **** <0.0001E2:esr2a+2b vs. E2:gper1 0.6493 0.4268 to 0.8718 Yes **** <0.0001
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Figure S5F:
Two-way ANOVA Ordinary Alpha 0.05 Source of Variation % of total variation P value P value summary Significant? Interaction 20.16 <0.0001 **** Yes GPER1 Factor 30.16 <0.0001 **** Yes Treatments Factor 15.09 <0.0001 **** Yes ANOVA table SS (Type III) DF MS F (DFn, DFd) P value Interaction 3.751 5 0.7502 F (5, 170) = 26.43 P<0.0001 GPER1 Factor 5.613 5 1.123 F (5, 170) = 39.55 P<0.0001 Treatments Factor 2.809 1 2.809 F (1, 170) = 98.98 P<0.0001 Residual 4.825 170 0.02838
Tukey's multiple comparisons test Mean Diff. 95.00% CI of diff. Significant? Summary Adjusted P Value
DMSO:gper1 +/+ vs. DMSO:gper1-/- 0.1459 -0.03293 to 0.3248 No ns 0.2335
DMSO:gper1 +/+ vs. E2:gper1 +/+ -0.6312 -0.8123 to -0.45 Yes **** <0.0001
DMSO:gper1 +/+ vs. E2:gper1-/- 0.01198 -0.1847 to 0.2086 No ns >0.9999
DMSO:gper1 +/+ vs. G1:gper1 +/+ -0.6046 -0.8104 to -0.3987 Yes **** <0.0001
DMSO:gper1 +/+ vs. G1:gper1-/- 0.1179 -0.1002 to 0.3361 No ns 0.8202
DMSO:gper1 +/+ vs. Rapa:gper1 +/+ 0.1946 0.008174 to 0.381 Yes * 0.0323
DMSO:gper1 +/+ vs. Rapa:gper1-/- 0.1042 -0.1311 to 0.3395 No ns 0.9473
DMSO:gper1 +/+ vs. Rapa+E2:gper1 +/+ 0.004425 -0.1965 to 0.2054 No ns >0.9999
DMSO:gper1 +/+ vs. Rapa+E2:gper1-/- 0.0678 -0.1217 to 0.2572 No ns 0.9894
DMSO:gper1 +/+ vs. Rapa+G1:gper1 +/+ 0.09971 -0.08669 to 0.2861 No ns 0.8303
DMSO:gper1 +/+ vs. Rapa+G1:gper1-/- 0.1612 -0.02517 to 0.3476 No ns 0.1627
DMSO:gper1-/- vs. E2:gper1 +/+ -0.7771 -0.956 to -0.5982 Yes **** <0.0001DMSO:gper1-/- vs. E2:gper1-/- -0.134 -0.3285 to 0.0606 No ns 0.4935
DMSO:gper1-/- vs. G1:gper1 +/+ -0.7505 -0.9544 to -0.5466 Yes **** <0.0001DMSO:gper1-/- vs. G1:gper1-/- -0.028 -0.2442 to 0.1882 No ns >0.9999
DMSO:gper1-/- vs. Rapa:gper1 +/+ 0.04863 -0.1356 to 0.2328 No ns 0.9993DMSO:gper1-/- vs. Rapa:gper1-/- -0.04177 -0.2753 to 0.1918 No ns >0.9999
DMSO:gper1-/- vs. Rapa+E2:gper1 +/+ -0.1415 -0.3404 to 0.0574 No ns 0.4405DMSO:gper1-/- vs. Rapa+E2:gper1-/- -0.07814 -0.2654 to 0.1091 No ns 0.9653
DMSO:gper1-/- vs. Rapa+G1:gper1 +/+ -0.04623 -0.2304 to 0.138 No ns 0.9995DMSO:gper1-/- vs. Rapa+G1:gper1-/- 0.01529 -0.1689 to 0.1995 No ns >0.9999
E2:gper1 +/+ vs. E2:gper1-/- 0.6431 0.4465 to 0.8398 Yes **** <0.0001
E2:gper1 +/+ vs. G1:gper1 +/+ 0.02659 -0.1793 to 0.2325 No ns >0.9999
E2:gper1 +/+ vs. G1:gper1-/- 0.7491 0.531 to 0.9672 Yes **** <0.0001
E2:gper1 +/+ vs. Rapa:gper1 +/+ 0.8257 0.6393 to 1.012 Yes **** <0.0001
E2:gper1 +/+ vs. Rapa:gper1-/- 0.7353 0.5 to 0.9706 Yes **** <0.0001
E2:gper1 +/+ vs. Rapa+E2:gper1 +/+ 0.6356 0.4346 to 0.8365 Yes **** <0.0001
E2:gper1 +/+ vs. Rapa+E2:gper1-/- 0.699 0.5095 to 0.8884 Yes **** <0.0001
E2:gper1 +/+ vs. Rapa+G1:gper1 +/+ 0.7309 0.5445 to 0.9173 Yes **** <0.0001
E2:gper1 +/+ vs. Rapa+G1:gper1-/- 0.7924 0.606 to 0.9788 Yes **** <0.0001
E2:gper1-/- vs. G1:gper1 +/+ -0.6165 -0.8362 to -0.3969 Yes **** <0.0001E2:gper1-/- vs. G1:gper1-/- 0.106 -0.1252 to 0.3371 No ns 0.9334
E2:gper1-/- vs. Rapa:gper1 +/+ 0.1826 -0.01891 to 0.3841 No ns 0.1168E2:gper1-/- vs. Rapa:gper1-/- 0.09219 -0.1553 to 0.3396 No ns 0.9854
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E2:gper1-/- vs. Rapa+E2:gper1 +/+ -0.007554 -0.2226 to 0.2075 No ns >0.9999E2:gper1-/- vs. Rapa+E2:gper1-/- 0.05582 -0.1485 to 0.2601 No ns 0.999
E2:gper1-/- vs. Rapa+G1:gper1 +/+ 0.08773 -0.1138 to 0.2892 No ns 0.9531E2:gper1-/- vs. Rapa+G1:gper1-/- 0.1493 -0.05225 to 0.3508 No ns 0.3758
G1:gper1 +/+ vs. G1:gper1-/- 0.7225 0.4834 to 0.9616 Yes **** <0.0001
G1:gper1 +/+ vs. Rapa:gper1 +/+ 0.7991 0.5886 to 1.01 Yes **** <0.0001
G1:gper1 +/+ vs. Rapa:gper1-/- 0.7087 0.4539 to 0.9636 Yes **** <0.0001
G1:gper1 +/+ vs. Rapa+E2:gper1 +/+ 0.609 0.3855 to 0.8325 Yes **** <0.0001
G1:gper1 +/+ vs. Rapa+E2:gper1-/- 0.6724 0.4591 to 0.8856 Yes **** <0.0001
G1:gper1 +/+ vs. Rapa+G1:gper1 +/+ 0.7043 0.4938 to 0.9148 Yes **** <0.0001
G1:gper1 +/+ vs. Rapa+G1:gper1-/- 0.7658 0.5553 to 0.9763 Yes **** <0.0001
G1:gper1-/- vs. Rapa:gper1 +/+ 0.07663 -0.1459 to 0.2991 No ns 0.9923G1:gper1-/- vs. Rapa:gper1-/- -0.01377 -0.2786 to 0.2511 No ns >0.9999
G1:gper1-/- vs. Rapa+E2:gper1 +/+ -0.1135 -0.3484 to 0.1213 No ns 0.9062G1:gper1-/- vs. Rapa+E2:gper1-/- -0.05014 -0.2752 to 0.1749 No ns 0.9999
G1:gper1-/- vs. Rapa+G1:gper1 +/+ -0.01823 -0.2407 to 0.2043 No ns >0.9999G1:gper1-/- vs. Rapa+G1:gper1-/- 0.04329 -0.1792 to 0.2658 No ns >0.9999
Rapa:gper1 +/+ vs. Rapa:gper1-/- -0.0904 -0.3298 to 0.149 No ns 0.9837
Rapa:gper1 +/+ vs. Rapa+E2:gper1 +/+ -0.1901 -0.3959 to 0.01556 No ns 0.1
Rapa:gper1 +/+ vs. Rapa+E2:gper1-/- -0.1268 -0.3212 to 0.0677 No ns 0.5804
Rapa:gper1 +/+ vs. Rapa+G1:gper1 +/+ -0.09486 -0.2864 to 0.09664 No ns 0.891
Rapa:gper1 +/+ vs. Rapa+G1:gper1-/- -0.03334 -0.2248 to 0.1582 No ns >0.9999
Rapa:gper1-/- vs. Rapa+E2:gper1 +/+ -0.09974 -0.3506 to 0.1511 No ns 0.9758Rapa:gper1-/- vs. Rapa+E2:gper1-/- -0.03637 -0.2781 to 0.2054 No ns >0.9999
Rapa:gper1-/- vs. Rapa+G1:gper1 +/+ -0.004459 -0.2438 to 0.2349 No ns >0.9999Rapa:gper1-/- vs. Rapa+G1:gper1-/- 0.05706 -0.1823 to 0.2964 No ns 0.9997
Rapa+E2:gper1 +/+ vs. Rapa+E2:gper1-/- 0.06337 -0.1451 to 0.2718 No ns 0.9974
Rapa+E2:gper1 +/+ vs. Rapa+G1:gper1 +/+ 0.09529 -0.1104 to 0.301 No ns 0.9286
Rapa+E2:gper1 +/+ vs. Rapa+G1:gper1-/- 0.1568 -0.0489 to 0.3625 No ns 0.3311
Rapa+E2:gper1-/- vs. Rapa+G1:gper1 +/+ 0.03191 -0.1626 to 0.2264 No ns >0.9999Rapa+E2:gper1-/- vs. Rapa+G1:gper1-/- 0.09343 -0.101 to 0.2879 No ns 0.9097
Rapa+G1:gper1 +/+ vs. Rapa+G1:gper1-/- 0.06152 -0.13 to 0.253 No ns 0.9957
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Figure S6A: Male
Two-way ANOVA Ordinary Alpha 0.05 Source of Variation % of total variation P value P value summary Significant? Interaction 18.47 <0.0001 **** Yes GPER1 Factor 51.16 <0.0001 **** Yes E2 Treatment Factor 22.5 <0.0001 **** Yes ANOVA table SS (Type III) DF MS F (DFn, DFd) P value Interaction 223.4 1 223.4 F (1, 30) = 39.61 P<0.0001 GPER1 Factor 618.8 1 618.8 F (1, 30) = 109.7 P<0.0001 E2 Treatment Factor 272.1 1 272.1 F (1, 30) = 48.25 P<0.0001 Residual 169.2 30 5.64
Tukey's multiple comparisons test Mean Diff. 95.00% CI of diff. Significant? Summary Adjusted P Value
DMSO:gper1 +/+ vs. DMSO:gper1 -/- 0.54 -2.348 to 3.428 No ns 0.9564
DMSO:gper1 +/+ vs. E2:gper1 +/+ -13.88 -17.06 to -10.69 Yes **** <0.0001
DMSO:gper1 +/+ vs. E2:gper1 -/- -2.92 -6.102 to 0.2623 No ns 0.0812
DMSO:gper1 -/- vs. E2:gper1 +/+ -14.42 -17.6 to -11.23 Yes **** <0.0001
DMSO:gper1 -/- vs. E2:gper1 -/- -3.46 -6.642 to -0.2777 Yes * 0.029
E2:gper1 +/+ vs. E2:gper1 -/- 10.96 7.505 to 14.41 Yes **** <0.0001
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Figure S6A: Female
Two-way ANOVA Ordinary Alpha 0.05 Source of Variation % of total variation P value P value summary Significant? Interaction 0.2994 0.6605 ns No GPER1 Factor 29.41 0.0001 *** Yes E2 Treatment Factor 28.21 0.0002 *** Yes ANOVA table SS (Type III) DF MS F (DFn, DFd) P value Interaction 4.86 1 4.86 F (1, 30) = 0.1968 P=0.6605 GPER1 Factor 477.3 1 477.3 F (1, 30) = 19.33 P=0.0001 E2 Treatment Factor 457.9 1 457.9 F (1, 30) = 18.54 P=0.0002 Residual 741 30 24.7
Figure S6B:
Tukey's multiple comparisons test Mean Diff. 95.00% CI of diff. Significant? Summary Adjusted P Value
DMSO:gper1 +/+ vs. DMSO:gper1 -/- 6.642 0.4332 to 12.85 Yes * 0.0325
DMSO:gper1 +/+ vs. E2:gper1 +/+ -8.323 -14.98 to -1.663 Yes ** 0.0099
DMSO:gper1 +/+ vs. E2:gper1 -/- -0.155 -6.565 to 6.255 No ns 0.9999
DMSO:gper1 -/- vs. E2:gper1 +/+ -14.97 -21.78 to -8.155 Yes **** <0.0001
DMSO:gper1 -/- vs. E2:gper1 -/- -6.797 -13.36 to -0.2309 Yes * 0.0403
E2:gper1 +/+ vs. E2:gper1 -/- 8.168 1.174 to 15.16 Yes * 0.0172
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Male
Two-way ANOVA Ordinary Alpha 0.05 Source of Variation % of total variation P value P value summary Significant? Interaction 8.594 0.0218 * Yes GPER1 Factor 1.425 0.3325 ns No E2 Treatment Factor 51.65 <0.0001 **** Yes ANOVA table SS (Type III) DF MS F (DFn, DFd) P value Interaction 15952 1 15952 F (1, 30) = 5.851 P=0.0218 GPER1 Factor 2646 1 2646 F (1, 30) = 0.9704 P=0.3325 E2 Treatment Factor 95882 1 95882 F (1, 30) = 35.17 P<0.0001 Residual 81793 30 2726
Tukey's multiple comparisons test Mean Diff. 95.00% CI of diff. Significant? Summary ted P Value
DMSO:gper1 +/+ vs. DMSO:gper1 -/- 63.89 0.3949 to 127.4 Yes * 0.0481
DMSO:gper1 +/+ vs. E2:gper1 +/+ -26.09 -96.06 to 43.88 No ns 0.7427
DMSO:gper1 +/+ vs. E2:gper1 -/- 125.8 55.86 to 195.8 Yes *** 0.0002
DMSO:gper1 -/- vs. E2:gper1 +/+ -89.98 -159.9 to -20.01 Yes ** 0.0077
DMSO:gper1 -/- vs. E2:gper1 -/- 61.94 -8.032 to 131.9 No ns 0.0974
E2:gper1 +/+ vs. E2:gper1 -/- 151.9 76.02 to 227.8 Yes **** <0.0001
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Figure S6B: Female
Two-way ANOVA Ordinary Alpha 0.05 Source of Variation % of total variation P value P value summary Significant? Interaction 1.526 0.2568 ns No GPER1 Factor 16.86 0.0006 *** Yes E2 Treatment Factor 41.54 <0.0001 **** Yes ANOVA table SS (Type III) DF MS F (DFn, DFd) P value Interaction 5242 1 5242 F (1, 30) = 1.337 P=0.2568 GPER1 Factor 57923 1 57923 F (1, 30) = 14.77 P=0.0006 E2 Treatment Factor 142701 1 142701 F (1, 30) = 36.38 P<0.0001 Residual 117666 30 3922
Tukey's multiple comparisons test Mean Diff. 95.00% CI of diff. Significant? Summary ted P Value
DMSO:gper1 +/+ vs. DMSO:gper1 -/- 155.8 77.53 to 234 Yes **** <0.0001
DMSO:gper1 +/+ vs. E2:gper1 +/+ 108.3 24.42 to 192.3 Yes ** 0.0074
DMSO:gper1 +/+ vs. E2:gper1 -/- 214 133.2 to 294.8 Yes **** <0.0001
DMSO:gper1 -/- vs. E2:gper1 +/+ -47.44 -133.3 to 38.38 No ns 0.4483
DMSO:gper1 -/- vs. E2:gper1 -/- 58.23 -24.52 to 141 No ns 0.244
E2:gper1 +/+ vs. E2:gper1 -/- 105.7 17.53 to 193.8 Yes * 0.014
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Figure S6C: Male
Two-way ANOVA Ordinary Alpha 0.05 Source of Variation % of total variation P value P value summary Significant? Interaction 2.14 0.2338 ns No GPER1 Factor 14.49 0.0036 ** Yes E2 Treatment Factor 35.5 <0.0001 **** Yes ANOVA table SS (Type III) DF MS F (DFn, DFd) P value Interaction 0.04501 1 0.04501 F (1, 30) = 1.476 P=0.2338 GPER1 Factor 0.3047 1 0.3047 F (1, 30) = 9.995 P=0.0036 E2 Treatment Factor 0.7465 1 0.7465 F (1, 30) = 24.48 P<0.0001 Residual 0.9146 30 0.03049
Tukey's multiple comparisons test Mean Diff. 95.00% CI of diff. Significant? Summary Adjusted P Value
DMSO:gper1 +/+ vs. DMSO:gper1 -/- 0.375 0.1627 to 0.5873 Yes *** 0.0002
DMSO:gper1 +/+ vs. E2:gper1 +/+ 0.2663 0.03231 to 0.5003 Yes * 0.0209
DMSO:gper1 +/+ vs. E2:gper1 -/- 0.4934 0.2595 to 0.7274 Yes **** <0.0001
DMSO:gper1 -/- vs. E2:gper1 +/+ -0.1087 -0.3427 to 0.1253 No ns 0.5926
DMSO:gper1 -/- vs. E2:gper1 -/- 0.1184 -0.1155 to 0.3524 No ns 0.5234
E2:gper1 +/+ vs. E2:gper1 -/- 0.2271 -0.02664 to 0.4809 No ns 0.0922
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Figure S6C: Female
Two-way ANOVA Ordinary Alpha 0.05 Source of Variation % of total variation P value P value summary Significant? Interaction 3.482 0.0251 * Yes GPER1 Factor 9.218 0.0006 *** Yes E2 Treatment Factor 67.83 <0.0001 **** Yes ANOVA table SS (Type III) DF MS F (DFn, DFd) P value Interaction 0.09375 1 0.09375 F (1, 30) = 5.561 P=0.0251 GPER1 Factor 0.2482 1 0.2482 F (1, 30) = 14.72 P=0.0006 E2 Treatment Factor 1.826 1 1.826 F (1, 30) = 108.3 P<0.0001 Residual 0.5057 30 0.01686
Tukey's multiple comparisons test Mean Diff. 95.00% CI of diff. Significant? Summary Adjusted P Value
DMSO:gper1 +/+ vs. DMSO:gper1 -/- 0.3617 0.1995 to 0.5239 Yes **** <0.0001
DMSO:gper1 +/+ vs. E2:gper1 +/+ 0.06643 -0.1076 to 0.2404 No ns 0.7287
DMSO:gper1 +/+ vs. E2:gper1 -/- 0.64 0.4725 to 0.8075 Yes **** <0.0001
DMSO:gper1 -/- vs. E2:gper1 +/+ -0.2952 -0.4732 to -0.1173 Yes *** 0.0005
DMSO:gper1 -/- vs. E2:gper1 -/- 0.2783 0.1068 to 0.4499 Yes *** 0.0007
E2:gper1 +/+ vs. E2:gper1 -/- 0.5736 0.3909 to 0.7563 Yes **** <0.0001
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Figure S6D: Male
Two-way ANOVA Ordinary Alpha 0.05 Source of Variation % of total variation P value P value summary Significant? Interaction 1.223 0.3885 ns No ANAS Factor 10.18 0.0152 * Yes GPER1 Factor 3.311 0.1583 ns No ANOVA table SS (Type III) DF MS F (DFn, DFd) P value Interaction 0.3103 1 0.3103 F (1, 54) = 0.7557 P=0.3885 ANAS Factor 2.584 1 2.584 F (1, 54) = 6.293 P=0.0152 GPER1 Factor 0.8402 1 0.8402 F (1, 54) = 2.046 P=0.1583 Residual 22.17 54 0.4106
Tukey's multiple comparisons test Mean Diff. 95.00% CI of diff. Significant? Summary Adjusted P Value
DMSO:gper1 +/+ vs. DMSO:gper1 -/- 0.3954 -0.2893 to 1.08 No ns 0.4266
DMSO:gper1 +/+ vs. ANAS:gper1 +/+ 0.5808 -0.1127 to 1.274 No ns 0.1308
DMSO:gper1 +/+ vs. ANAS:gper1 -/- 0.6772 0.0003279 to 1.354 Yes * 0.0498
DMSO:gper1 -/- vs. ANAS:gper1 +/+ 0.1854 -0.4251 to 0.7958 No ns 0.8518
DMSO:gper1 -/- vs. ANAS:gper1 -/- 0.2818 -0.3098 to 0.8735 No ns 0.5902
ANAS:gper1 +/+ vs. ANAS:gper1 -/- 0.09649 -0.5052 to 0.6982 No ns 0.9739
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Figure S6D:
Female
Two-way ANOVA Ordinary Alpha 0.05 Source of Variation % of total variation P value P value summary Significant? Interaction 13.15 0.0092 ** Yes ANAS Factor 5.314 0.0907 ns No GPER1 Factor 0.8928 0.4824 ns No ANOVA table SS (Type III) DF MS F (DFn, DFd) P value Interaction 2.435 1 2.435 F (1, 46) = 7.39 P=0.0092 ANAS Factor 0.9838 1 0.9838 F (1, 46) = 2.986 P=0.0907 GPER1 Factor 0.1653 1 0.1653 F (1, 46) = 0.5016 P=0.4824 Residual 15.16 46 0.3295
Tukey's multiple comparisons test Mean Diff. 95.00% CI of diff. Significant? Summary Adjusted P Value
DMSO:gper1+/+ vs. DMSO:gper1 -/- 0.5617 -0.04022 to 1.164 No ns 0.0753DMSO:gper1+/+ vs. ANAS:gper1+/+ 0.7289 0.07372 to 1.384 Yes * 0.0238
DMSO:gper1+/+ vs. ANAS:gper1 -/- 0.3993 -0.2026 to 1.001 No ns 0.3014
DMSO:gper1 -/- vs. ANAS:gper1+/+ 0.1671 -0.4664 to 0.8007 No ns 0.8953
DMSO:gper1 -/- vs. ANAS:gper1 -/- -0.1624 -0.7407 to 0.4159 No ns 0.8769
ANAS:gper1+/+ vs. ANAS:gper1 -/- -0.3295 -0.963 to 0.304 No ns 0.5141
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Table S1. PCR primers.
Gene Forward Primer (5'--- 3') Reverse Primer (5'- -- 3') LTR (5'--- 3')