Regulation of myeloid ecotropic viral integration site 1 and its expression in normal and abnormal endometrium  Linli Hu, M.D., Haixia Li, M.D., CindyTzu-Ling Huang, Ph.D., Hong Chen, M.D., Guijin Zhu, M.D., Kun Qian, M.D.  Fertility and Sterility  Volume 102, Issue 3, Pages 856-863.e2 (September 2014) DOI: 10.1016/j.fertnstert.2014.05.036 Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 1 MEIS1 expression in the human endometrium. Endometrium samples represent the 6 developmental phases of the reproductive cycle and decidua. (A) MEIS1 expression appears in the stroma and glands from early-proliferative (P1), midproliferative (P2), late-proliferative (P3), early-secretory (S1), midsecretory (S2), and late-secretory phases (S3); and decidua (D). Photomicrographs taken at ×400 magnification. (B) Western blot analysis of MEIS1 expression in endometrium during the menstrual cycle and decidua. Amplification of ß-actin was performed as a control. A representative western blot image is shown. MEIS1 protein expression was analyzed by densitometry and normalized to ß-actin values as described in the Materials and Methods section. Western blot analysis and quantification was repeated at least 3 times for each sample, and 10 different samples were analyzed. Error bars are standard error of the mean (SEM). *Statistical significance (P<.05). Fertility and Sterility 2014 102, 856-863.e2DOI: (10.1016/j.fertnstert.2014.05.036) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Expression of MEIS1 in primary endometrial stromal cells (ESCs) and Ishikawa cells in response to sex steroids. (A) Phase contrast images of ESCs. (B) Immunostaining of MEIS1 expression in ESCs. (C) Western blot analysis of MEIS1 expression in ESCs. Cells were treated with 10 × 10−8 M E2 (E), 10 × 10−8 M MPA (P), or both hormones for 24 hours. (D) Phase contrast images of Ishikawa cells. (E) Immunostaining of MEIS1 expression in Ishikawa cells. (F) Western blot analysis of MEIS1 expression in Ishikawa cells. Cells were treated as ESCs. MEIS1 protein expression was analyzed by densitometry and normalized to ß-actin values. Western blot analysis and quantification was repeated at least 3 times for each sample, and 3 different samples were analyzed. Error bars are SEM. *Statistical significance (P<.05). Fertility and Sterility 2014 102, 856-863.e2DOI: (10.1016/j.fertnstert.2014.05.036) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 3 MEIS1 expression in the eutopic endometrium and ectopic endometrium in endometriosis. (A) Immunohistochemistry images of MEIS1 expression in ectopic endometrium (Ecto), eutopic endometrium (Euto), and normal endometrium (Nor) during midsecretory phases. Photomicrographs taken at ×400 magnification. (B) Western blot analysis of MEIS1 expression in eutopic endometrium and normal endometrium during midsecretory phases. Western blot analysis and quantification was repeated at least 3 times for each sample, and 10 different samples were analyzed. Error bars are SEM. *Statistical significance (P<.05). Fertility and Sterility 2014 102, 856-863.e2DOI: (10.1016/j.fertnstert.2014.05.036) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 4 A proposed model of MEIS1 expression regulated by sex steroids and genes for uterine receptivity (GsUR) regulated by HOXA10, MEIS1, or a HOXA10-MEIS1 heterodimer. ER = E receptor; PR = P receptor. Fertility and Sterility 2014 102, 856-863.e2DOI: (10.1016/j.fertnstert.2014.05.036) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Expression of MEIS1 in mouse uteri during early pregnancy. (A) MEIS1 expression location during early pregnancy from D1 to D8. Photomicrographs taken at ×40 magnification. The arrows indicate the location of embryos. Le = luminal epithelium; myo = myometrium; m = mesometrial pole; am = antimesometrial pole; Dc = decidua. (B) Western blot analysis of MEIS1 expression during early pregnancy from D1 to D8. Each group consisted of 10 mice, and the experiments were repeated at least 3 times for each mouse. Error bars are SEM. *Statistical significance (P<.05). D = day. Fertility and Sterility 2014 102, 856-863.e2DOI: (10.1016/j.fertnstert.2014.05.036) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 2 Uterine expression of MEIS1 in oophorectomized mice after steroid hormone treatment. (A) Quantitative RT-PCR analysis of MEIS1 mRNA in uteri of oophorectomized mice treated with C (control), E2 (100 ng/mouse), MPA (2 mg/mouse), or E2+MPA. The mice were euthanized at 12 hours posttreatment. (B) Immunostaining of MEIS1 expression in uteri of oophorectomized mice. MEIS1 expression appears in the stroma and glands after treatment with E2, MPA, or both. (C) Western blot analysis of MEIS1 expression in uteri of oophorectomized mice treated with P and/or E for the indicated time. The quantification was repeated at least 3 times for each sample, and 10 different samples were analyzed. Error bars are SEM. *Statistical significance (P<.05). Fertility and Sterility 2014 102, 856-863.e2DOI: (10.1016/j.fertnstert.2014.05.036) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions