1. 2-Methoxyestradiol Inhibits Hypoxia-Inducible Factor-1α and Suppresses Growth of Lesions in a Mouse Model of Endometriosis 
Christian M. Becker, Nadine Rohwer, Tae Funakoshi, Thorsten Cramer, Wanja Bernhardt, Amy Birsner, Judah Folkman, Robert J. D'Amato 
The American Journal of Pathology 
Volume 172, Issue 2, Pages (February 2008)
DOI: /ajpath
Copyright © 2008 American Society for Investigative Pathology Terms and Conditions

2. Figure 1
A: Hypoxyprobe staining. Representative murine endometriotic tissue removed at different time points after transplantation. D: 1 hour. E: 4 hours. F: 24 hours. G: 48 hours. H: 1 week. I: 6 months. Each mouse was injected with pimonidazole hydrochloride (hypoxyprobe-1) 30 minutes before removal. Eutopic uterine lesions from the same mice were used as negative controls (A–C). Staining with hypoxyprobe antibody and counterstaining with hematoxylin. Control endometriotic tissue (4 hours; J) stained with an isotype-matched irrelevant murine IgG1 monoclonal antibody directed against Shigela toxin.
The American Journal of Pathology  , DOI: ( /ajpath )
Copyright © 2008 American Society for Investigative Pathology Terms and Conditions

3. Figure 2
A: Immunohistochemistry staining of endometriosis-like lesions with anti-HIF-1α antibody. Counterstaining with hematoxylin. Increasing immunoreactivity (brown color) in glandular epithelial and stromal cells (arrows) over time (0, 1, 3, and 6 hours). Control (6 hours) stained with nonspecific rabbit IgG antibody. B: Western blot of endometriotic tissue stained with ant-HIF-1α antibody from different time points after transplantation. Loading control of same Western blot stained with anti-β-actin antibody. C: Real-time RT PCR of endometriosis-like lesions at different time points using primers for HIF-1α-dependent genes (VEGF, PGK, and Glut-1). Bars indicate SEM. D: Semiquantitative measurement of Western blot for HIF-1α as seen in B. Error bars indicate SEM. E: Endometriosis-like lesion from a green fluorescent protein+/+ mouse on the peritoneal wall of a wild-type recipient. Typical growth of blood vessels into the endometriosis-like lesion.
The American Journal of Pathology  , DOI: ( /ajpath )
Copyright © 2008 American Society for Investigative Pathology Terms and Conditions

4. Figure 3
Systemic 2-methoxyestradiol inhibits growth of endometriosis-like lesions. Endometriosis-like lesions were measured in two perpendicular diameters (D1 and D2) with a caliper, and CSA was calculated using the formula of an ellipse (D1 × D2 × π/4) as previously described.20,21,24A: Mean CSA of endometriosis-like lesions (n = 7 lesions) of mice treated with vehicle (▪) or 2-methoxyestradiol (□) (n = 6/group). B: Mean CSA after 4 weeks of oral treatment with different doses of 2-methoxyestradiol. Bars indicate the SEM.
The American Journal of Pathology  , DOI: ( /ajpath )
Copyright © 2008 American Society for Investigative Pathology Terms and Conditions

5. Figure 4
Systemic 2-methoxyestradiol treatment reduces expression of HIF-1α target genes. Results of a representative experiment using real-time RT-PCR of endometriosis-like lesions removed after different time intervals after autotransplantation. Mice had been treated systemically with 2-methoxyestradiol before the induction of endometriosis. Lesions were removed at different time points (baseline, 1 hour, 3 hours, 24 hours, 72 hours, and 1 week) and tested for mRNA expression of VEGF (A), PGK (B), and Glut-1 (C). Bars indicate SEM.
The American Journal of Pathology  , DOI: ( /ajpath )
Copyright © 2008 American Society for Investigative Pathology Terms and Conditions

6. Figure 5
A: Western blot of endometriotic tissue samples at baseline (0 hours) and 6 hours. Lewis lung carcinoma is used as positive control in equivalent concentration. Staining with anti-HIF-1α antibody and anti-β actin as loading control. B: A modified Miles assay shows that systemic 2-methoxyestradiol treatment inhibits vascular permeability. Mice were pretreated orally with tap water (control), vehicle, and 2-methoxyestradiol for 5 days and then anesthetized and injected with Evans blue (i.v.). After 10 minutes, mice were given intradermal injections of PBS (50 μl; top two spots) and VEGF/vascular permeability factor (50 ng in 50 μl; bottom two spots), and mice were euthanized after 20 minutes. Reduced extravasation of dye in the skin of mice treated with 2-methoxyestradiol. C: Dye from excised skin was extracted with formamide for 5 days, and dye contents were measured at 620 nm. Data are expressed as mean ± SEM.
The American Journal of Pathology  , DOI: ( /ajpath )
Copyright © 2008 American Society for Investigative Pathology Terms and Conditions