G protein–coupled estrogen receptor 1 agonist G-1 induces cell cycle arrest in the mitotic phase, leading to apoptosis in endometriosis Taisuke Mori,

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G protein–coupled estrogen receptor 1 agonist G-1 induces cell cycle arrest in the mitotic phase, leading to apoptosis in endometriosis Taisuke Mori, M.D., Ph.D., Fumitake Ito, M.D., Hiroshi Matsushima, M.D., Osamu Takaoka, M.D., Yukiko Tanaka, M.D., Akemi Koshiba, M.D., Izumi Kusuki, M.D., Ph.D., Jo Kitawaki, M.D., Ph.D. Fertility and Sterility Volume 103, Issue 5, Pages 1228-1235.e1 (May 2015) DOI: 10.1016/j.fertnstert.2015.01.026 Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 1 (A) Effects of G-1 on the proliferation of ESCs. (B) Representative morphologic images of ESCs treated with the indicated concentrations of G-1. (C) Effects of GPER antagonism, MEK inhibition, and PI3K pathway inhibition or (D) GPER knockdown on the effects of G-1. (E, F) Expression of GPER protein in ESCs at (E) 48 hours after transfection with siRNA or (F) 96 hours after treatment with G-1 (10 μM). The results are expressed as a percentage of the control values and presented as means ± SEMs for at least six independent experiments. ∗ P<.05, ∗∗ P<.01 versus the control. Fertility and Sterility 2015 103, 1228-1235.e1DOI: (10.1016/j.fertnstert.2015.01.026) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Effects of G-1 on the cell cycle and survival in ESCs. (A, B) Flow cytometry analysis of the effects of G-1 on (A) the ESC cell cycle distribution and (B) the sub-G1 population. (C) The cytotoxic and caspase activation effects of G-1 (10 μM) in ESCs. Values are presented as means ± SEMs for at least six independent experiments. ∗ P<.05, ∗∗ P<.01 versus the control. DMSO = dimethyl sulfoxide. Fertility and Sterility 2015 103, 1228-1235.e1DOI: (10.1016/j.fertnstert.2015.01.026) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Endometriotic stromal cell analysis by immunofluorescence cytochemistry. (A, B) Representative images of (A) the cell cycle phases and (B) their proportion with or without G-1 treatment. Endometriotic stromal cells were stained with antibodies against α-tubulin (green) or phospho-histone H3 (Ser10) (magenta; pseudocolor) after a 96-hour incubation with G-1 (10 μM). The nuclei were counterstained with PI (red). Data are presented as means ± SEMs for five independent experiments. ∗ P<.05, ∗∗ P<.01 versus the control. Fertility and Sterility 2015 103, 1228-1235.e1DOI: (10.1016/j.fertnstert.2015.01.026) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 4 (A, B) Effect of G-1 on protein expression patterns in (A) the cell cycle and (B) survival of endometriotic stromal cells. After a 96-hour treatment with G-1 (10 μM), the levels of cell M-phase promoting factor-related proteins (phospho-histone H3, cyclin B1, phospho-cdc2, cdc2, phospho-cdc25c, cdc25c, phospho-wee1, and wee1) and apoptosis-related proteins (caspase 3, survivin, XIAP, p21, and Bcl-2) were examined by Western blot analysis. Representative results of at least six independent experiments are shown. Fertility and Sterility 2015 103, 1228-1235.e1DOI: (10.1016/j.fertnstert.2015.01.026) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Western blot analysis of the effects of G-1 on the proteins involved in G protein-coupled estrogen receptor signaling. (A) Phosphorylated and total protein expression of ERK in ESCs treated with G-1 (10 μM) for 30 minutes, G-15 (10 μM) for 30 minutes, U0126 (10 μM) for 30 minutes, or a combination of 10 μM G-1 and 10 μM G-15 (G-1 added 30 minutes after G-15) or 10 μM G-1 and 10 μM U0126 (G-1 added 30 minutes after U0126). (B) Phosphorylated and total Akt levels in ESCs treated with G-1 (10 μM) for 30 minutes, G-15 (10 μM) for 30 minutes, LY294002 (10 μM) for 30 minutes, or a combination of 10 μM G-1 and 10 μM G-15 (G-1 added 30 minutes after G-15) or 10 μM G-1 and 10 μM LY294002 (G-1 added 30 minutes after LY294002). Representative results of at least six separate experiments are shown. Fertility and Sterility 2015 103, 1228-1235.e1DOI: (10.1016/j.fertnstert.2015.01.026) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions