Progressive development of endometriosis and its hindrance by anti-platelet treatment in mice with induced endometriosis  Qi Zhang, Xishi Liu, Sun-Wei Guo  Reproductive BioMedicine Online  Volume 34, Issue 2, Pages 124-136 (February 2017) DOI: 10.1016/j.rbmo.2016.11.006 Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions

Figure 1 The kinetic changes of platelet activation rate in peripheral blood samples (A), lesion weight (B), and hotplate latency (C) in mice treated with Tanshinone IIA (blue lines) and without (red lines). The data are shown in means and standard deviations. *: P < 0.05; NS = not statistically significant (by Wilcoxon rank test). n = 4–5 at any time point for each group. Reproductive BioMedicine Online 2017 34, 124-136DOI: (10.1016/j.rbmo.2016.11.006) Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions

Figure 2 Representative immunostaining staining results in ectopic lesions in mice treated with and without Tanshinone IIA (TAN) during the progression of endometriosis (Part I). TAN: mice treated with TAN; control (CTL): mice without treatment of TAN. Different columns represent tissue samples harvested at different time points (after induction of endometriosis). The level of CD41 staining reflected the aggregation of platelets, and mostly seen in cytoplasmic components in stromal cells. E-cadherin staining was seen mainly in cytoplasmic and membranous components in epithelial cells. Transforming growth factor-β1 (TGF-β1) staining was seen primarily in glandular epithelial cells and was localized in the cytoplasm. The phosphorylated Smad3 (p-Smad3) staining was seen in nuclei as well as cytoplasm in endometriotic epithelial cells. The p-Smad3 staining results for week 1 were unavailable since the tissue sample sections were run out. Scale bar = 50 µm. Reproductive BioMedicine Online 2017 34, 124-136DOI: (10.1016/j.rbmo.2016.11.006) Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions

Figure 3 Representative immunostaining staining results in ectopic lesions in mice treated with and without Tanshinone IIA (TAN) during the progression of endometriosis (Part II). TAN: mice treated with TAN; control (CTL): mice without treatment of TAN. Different columns represent tissue samples harvested at different time points (after induction of endometriosis). Collagen I and α-smooth muscle actin (α-SMA) staining was seen mostly in the stromal component of the ectopic lesions. Connective tissue growth factor (CCN2 or CTGF) staining was seen in cytoplasmic and extracellular matrix components in both glandular epithelial and stromal cells. Lysyl oxidase (LOX) staining was seen primarily in glandular epithelial cells and was localized in the cytoplasm. Scale bar = 50 µm. Reproductive BioMedicine Online 2017 34, 124-136DOI: (10.1016/j.rbmo.2016.11.006) Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions

Figure 4 Changes in platelet aggregation shown by CD41 staining (A), Transforming growth factor-β1 (TGF-β1) staining (B), phosphorylated Smad3 (p-Smad3) staining (C), E-cadherin staining (D), CCN2 staining (E), α-smooth muscle actin (α-SMA) staining (F), collagen I staining (G), lysyl oxidase (LOX) staining (H), desmin staining (I), and smooth muscle, myosin heavy chain (SM-MHC) staining (J), and the proportion of fibrotic content (K) in ectopic endometrium from treated with Tanshinone IIA (blue line) and without (red line). The data are shown in means and standard deviations. *: P < 0.05; **: P < 0.01; NS = not statistically significant (Wilcoxon rank test). n = 4–5 at any time point for each group. Reproductive BioMedicine Online 2017 34, 124-136DOI: (10.1016/j.rbmo.2016.11.006) Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions

Figure 5 Representative immunostaining of desmin and SM-MHC and Masson trichrome staining in ectopic lesions in mice treated with and without Tanshinone IIA (TAN) during the progression of endometriosis. TAN: mice treated with TAN; control (CTL): mice without treatment of TAN. Different columns represent tissue samples harvested at different time points (after induction of endometriosis). For desmin and SM-MHC, staining of ectopic endometrium harvested 6 weeks after induction was not performed since all sections were exhausted. Desmin and SM-MHC staining was seen mostly in the stromal component of the ectopic lesions. In Masson trichrome staining, the collagen fibres in ectopic endometrium were stained in blue. Scale bar = 50 µm. SM-MHC = smooth muscle, myosin heavy chain. Reproductive BioMedicine Online 2017 34, 124-136DOI: (10.1016/j.rbmo.2016.11.006) Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions

Figure 6 (A) Hierarchical clustering heatmap of immunoreactivity measurements and mice in the control group. The heatmap was organized by clustering both mice (by rows) and immunoreacivity measurements (by columns). The red colour represents the minimal values while the bright yellow colour represents the maximal values. The white colour strips represent missing values. The numeric on the right-hand side represents which time points (weeks after the induction of endometriosis) when the tissue samples were harvested. The labels in the bottom are the names of the immunoreactivity measurements. The colour strip on the left: The pink colour of increasing darkness indicates tissue samples taken from mice with more advanced endometriosis. The numeric on the rightmost panel represents which time points (weeks after the induction of endometriosis, i.e. age of lesions) when the tissue samples were harvested. PLT Ag: The extent of platelet aggregation; % aPLT: The platelet activation rate in the peripheral blood sample. (B) Results of multidimensional scaling analysis using the E-cadherin and α-smooth muscle actin (α-SMA) immunostaining levels and the extent of fibrosis in ectopic endometrium. Each ellipse indicates one group. The numbers within the ellipses indicate which time points (i.e. weeks after the induction of endometriosis) when the tissue samples were harvested, that is, the age of the lesion tissues. Each number represents a tissue sample from one mouse. Different colours of the tissue samples are used to highlight the age of the lesions, which are consistent with the colours used in panel (A). Reproductive BioMedicine Online 2017 34, 124-136DOI: (10.1016/j.rbmo.2016.11.006) Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions