(A) Heatmap representing normalized genome-wide DNA binding by ERα via ChIP sequencing analyses centered according to peak summits for each treatment group and compared to each other group. (B) …
PCA plot of genome-wide ERα binding profiles by ChIP sequencing analyses. U2OS-ERα cells treated with low dose (10 nM) 17α-E2, high dose (100 nM) 17α-E2, or 17β-E2 (10 nM) induced binding in common …
(A) Percent change in mass (mean ± SEM, two-way repeated measures ANOVA with Holm-Sidak post-hoc; *p<0.05, **p<0.005 between WT HFD and WT HFD+17α), (B) Mass at baseline (week 0; solid) and week 14 …
(A) Fasting glucose (mean ± SEM, two-way repeated measures ANOVA), (B) Fasting insulin (mean ± SEM, two-way repeated measures ANOVA with Holm-Sidak post-hoc; *p<0.05, **p<0.005), and (C) …
(A) Glucose tolerance testing (GTT; 1 mg/kg), normalized to baseline (min 0) (mean ± SEM, two-way repeated measures ANOVA with Holm-Sidak post-hoc; *p<0.05 between WT HFD and WT HFD+17α), and (B) …
(A) Mass at baseline (week 0; solid) and week 4 (striped) (mean ± SEM, two-way repeated measures ANOVA), (B) Fat mass at baseline (week 0; solid) and week 4 (striped) (mean ± SEM, two-way repeated …
(A) Liver mass (mean ± SEM, two-way ANOVA with Holm-Sidak post-hoc; *p<0.05), (B) Representative liver oil-red-O staining, (C) Liver fatty acids (mean ± SEM, two-way ANOVA with Holm-Sidak post-hoc; …
(A) Quantification of oil-red-O lipid staining in liver sections from WT and ERα KO mice provided 45% HFD (TestDiet 58V8)±17α-E2 (14.4ppm) for 14 weeks (mean ± SEM, two-way ANOVA with Holm-Sidak …
Relative molar % of fatty acids in the liver from WT and ERα KO mice provided 45% HFD (TestDiet 58V8)±17α-E2 (14.4ppm) for 14 weeks (mean ± SEM, Benjamini–Hochberg multiple testing correction, …
(A) Representative liver Masson’s trichrome staining for collagen and (B) Liver transcriptional markers of fibrosis in WT and ERα KO mice provided 45% HFD (TestDiet 58V8)±17α-E2 (14.4ppm) for 14 …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Genetic reagent (M. musculus) | B6N(Cg)-Esr1tm4.2Ksk/J | The Jackson Laboratory | Stock No:026176; RRID:IMSR_JAX:026176 | ERα (Esr1) KO mice |
Cell line (Homo sapien) | U2OS Cells | ATCC | HTB-96; RRID:CVCL_0042 | PMID:15802376 PMID:14505348 |
Antibody | anti-FLAG M2 (Mouse monoclonal) | Sigma-Aldrich | F1804 | IP: 1 uL per pull-down (1 mg/mL) |
Commercial assay or kit | Protein G Dynabeads | Applied Biosystems/Thermofisher Scientific | 10003D | IP: 30 uL per IP |
Chemical compound, drug | 17α-E2 | Steraloids, Inc | E0870-000 | |
Chemical compound, drug | Novolin R 100 U/ml | Novolin | 2mU/g | |
Other (diet) | Chow; TestDiet 58YP | TestDiet | TestDiet 58YP | |
Other (diet) | HFD; TestDiet 58V8 | TestDiet | TestDiet 58V8 | HFD 45% by kcal |
Other (diet) | HFD; TestDiet 58Y1 | TestDiet | TestDiet 58Y1 | HFD 60% by kcal |
Commercial assay or kit | Mouse Ultrasensitive Insulin ELISA | ALPCO | Cat# 80-INSMSU-E01; RRID:AB_2792981 | |
Commercial assay or kit | Free Glycerol Agent | Sigma-Aldrich | Sigma F6428 | |
Commercial assay or kit | Triglyceride Reagent | Sigma-Aldrich | Sigma F6428 | |
Commercial assay or kit | Glycerol Standard | Sigma-Aldrich | Sigma G1394 | |
Antibody | anti-pS473 AKT (Rabbit polyclonal) | Abcam | Cat# ab81283; RRID:AB_2224551 | WB: (1:3000) |
Antibody | Anti-pan-AKT (Rabbit polyclonal) | Abcam | Cat# ab179463; RRID:AB_2810977 | WB (1:10000) |
Antibody | Anti-pS256 FOX01 (Rabbit polyclonal) | Abcam | Cat# ab131339; RRID:AB_11159015 | WB (1:1000) |
Antibody | Anti-FOX01a (Rabbit polyclonal) | Abcam | Cat# ab52857; RRID:AB_869817 | WB (1:1000) |
Antibody | Anti-GAPDH (Rabbit polyclonal) | Abcam | Cat# ab9485; RRID:AB_307275 | WB (1:2500) |
Antibody | Anti-Rabbit IgG, IRDye 800 CW | LI-COR | Cat# 926–32211; RRID:AB_621843 | WB (1:15000) |
Commercial assay or kit | TaqMan Gene Expression Master Mix | Applied Biosystems/Thermofisher Scientific | 4369542 | |
Sequenced-based reagent | qPCR primer Mmp1 | Integrated DNA Technologies | Mm.PT.58.42286812 Ref Seq: NM_008607(1) | Exon 5–6 |
Sequenced-based reagent | qPCR primer Mmp12 | Integrated DNA Technologies | Mm.PT.58.31615472 Ref Seq: NM_008605(1) | Exon 8–9 |
Sequenced-based reagent | qPCR primer Ccl2 | Integrated DNA Technologies | Mm.PT.58.42151692 Ref Seq: NM_011333(1) | Exon 1–3 |
Sequenced-based reagent | qPCR primer Srebf1 | Integrated DNA Technologies | Mm.PT.58.8508227 Ref Seq: NM_011480(1) | Exon 1–2 |
Sequenced-based reagent | qPCR primer Pck1 | Integrated DNA Technologies | Mm.PT.58.11992693 Ref Seq: NM_011044(1) | Exon 3–4 |
Sequenced-based reagent | qPCR primer Cdkn1a | Integrated DNA Technologies | Mm.PT.58.17125846 Ref Seq: NM_007669(1) | Exon 2–3 |
Sequenced-based reagent | qPCR primer Pparα | Integrated DNA Technologies | Mm.PT.58.9374886 Ref Seq: NM_001113418(2) | Exon 8–9 |
Sequenced-based reagent | qPCR primer Cxcl1 | Integrated DNA Technologies | Mm.PT.58.42076891 Ref Seq: NM_008176(1) | Exon 2–4 |
Sequenced-based reagent | qPCR primer Col1a1 | Integrated DNA Technologies | Mm.PT.58.7562513 Ref Seq: M_007742(1) | Exon 1–2 |
Sequenced-based reagent | qPCR primer Tnfrsf1a | Integrated DNA Technologies | Mm.PT.58.28810479 Ref Seq: NM_011609(1) | Exon 5–7 |
Software, algorithm | SigmaPlot 12.5 | Systat Software | RRID:SCR_003210 | statistical analyses |
Software, algorithm | ImageJ | ImageJ | RRID:SCR_003070 | histological quantification |
Software, algorithm | Image Studio | LI-COR | RRID:SCR_015795 | western blot quantification |
Software, algorithm | RStudio | GenomicAlignments DiffBind DESeq2 GenomicRanges | RRID:SCR_000432 | Peak Calling Differential expression Differential binding |
Software, algorithm | Bowtie2 MACS2 Bedtools Samtools Picard-tools Trimmomatic | Bowtie2 MACS2 Bedtools Samtools Picard-tools Trimmomatic | Alignment, Peak Calling, trimming, duplicate identification |
Pairwise statistical comparisons of ERα binding.
Negative binomial regression Wald post-hoc comparison test, FDR < 0.05. n = 3/group.
ERα binding motif analysis.
Motif analysis was performed using HOMER with standard settings with the significance threshold set to FDR corrected p<0.05. Peak regions called for each treatment group were analyzed to identify enriched motifs relative to the entire genome. Hypergeometric test was used to test enrichment. Only motifs with FDR corrected p<0.05 were reported as significant. For pairwise differential motif enrichment or depletion across experimental groups, we utilized the hypergeometric test by using the number of sequences with motif from each group and total number of peaks as total sample size. Motifs that appear in less than five sequences between both test groups were removed. Benjamini-Hochberg multiple testing correction was utilized to control for false discovery rate (FDR < 0.05).
Circulating eicosanoid levels (pmol/ml).
17α-E2 mildly alters the circulating eicosanoid profile in obese middle-aged male mice. WT mice were provided 45% HFD (TestDiet 58V8)±17α-E2 (14.4ppm) for 14 weeks. Age-matched, male WT, chow-fed (TestDiet 58YP) mice were also evaluated as a normal-weight reference group. All data are presented as mean ± SEM and were analyzed by Student’s t-test with the WT Chow group being excluded from statistical comparisons. n = 5–7.