2-Methoxyestradiol causes functional repression of transforming growth factor β3 signaling by ameliorating Smad and non-Smad signaling pathways in immortalized uterine fibroid cells  Salama A. Salama, Ph.D., Concepcion R. Diaz-Arrastia, M.D., Gokhan S. Kilic, M.D., Marwa W. Kamel, M.Sc.  Fertility and Sterility  Volume 98, Issue 1, Pages 178-184.e1 (July 2012) DOI: 10.1016/j.fertnstert.2012.04.002 Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Transforming growth factor (TGF) β3 activates Smad and non-Smad PI3K/Akt/mTOR signaling pathways in immortalized human uterine fibroid smooth muscle (huLM) cells. Serum-starved cells were pretreated with Smad3-specific inhibitor SIS3 and/or PI3K/Akt inhibitor LY294002 for 1 hour before stimulation with TGF-β3. Thirty minutes after treatment with TGF-β3, the cell lysates were used for measuring the expression of: (A) pSmad2 (Ser 465/467), Smad3, and pSmad3 (Ser 423/425); and (B) Akt, pAkt (Ser 473), S6 ribosomal protein, and pS6 (Ser 235/236) by Western blot. (C) Serum-starved huLM cells were treated with TGF-β3 in the presence or absence of SIS3 and/or LY294002 for 24 hours. mRNA levels of collagen (Col) I(αI), Col III(αI), connective tissue growth factor (CTGF), and plasminogen activator inhibitor (PAI) 1 were measured by real-time reverse-transcription polymerase chain reaction (RT-PCR) and normalized to GAPDH. Results are reported as fold change compared with cells treated with vehicle only and represented as the mean ± SEM of three independent experiments. ∗A: significantly higher compared with the vehicle-treated control; ∗B: significantly higher than the control cells but lower than cells treated with TGF-β3 alone; ∗C: significantly lower than cells treated with TGF-β3 alone or in combination with SIS3 or LY294002. Fertility and Sterility 2012 98, 178-184.e1DOI: (10.1016/j.fertnstert.2012.04.002) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 2 2-Methoxyestradiol (2ME2) ameliorates TGF-β3–induced expression of profibrotic genes in huLM cells. (A) Serum-starved huLM cells were treated with TGF-β3 and/or 2ME2 for 24 hours. The mRNA levels of Col I(αI), Col III(αI), CTGF, and PAI-1 genes were measured by real-time RT-PCR and normalized to GAPDH. Results are reported as fold change compared with cells treated with vehicle only and represented as the mean ± SEM out of 3 independent experiments. ∗Significantly lower than control; ∗a: significantly higher than control; ∗b: significantly lower than TGF-β3–treated cells. (B) Serum-starved huLM cells were stimulated with TGF-β3 and/or 2ME2 for 48 hours. Cell lysates were collected and the expression level of α-smooth muscle actin (α-SMA), CTGF, and PAI-1 proteins were determined by immunoblotting. Other abbreviations as in Figure 1. Fertility and Sterility 2012 98, 178-184.e1DOI: (10.1016/j.fertnstert.2012.04.002) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Effect of 2ME2 on TGF-β3–induced phosphorylation of Smad2 and Smad3 at the carboxy termini (A) and linker regions (B) in huLM cells. The cells were serum starved for 24 hours and then treated with TGF-β3 with or without 2ME2. The cell lysates were analyzed by Western blotting with the use of antibodies against C-terminal pSmad2 (pSer465/467), C-terminal pSmad3 (pSer423/425), linker-region pSmad2 (pSer245/Ser250/Ser255), linker-region pSmad3 (pSer-204), Smad2, or Smad3. Western blot with β-actin was used as loading control. Abbreviations as in Figures 1 and 2. Fertility and Sterility 2012 98, 178-184.e1DOI: (10.1016/j.fertnstert.2012.04.002) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Effect of 2ME2 on TGF-β3–induced nuclear translocation of pSmad2 and pSmad3 in huLM cells. (A) Cytoplasmic and nuclear fractions were prepared from huLM cells treated with TGF-β3 with or without 2ME2 as indicated. Equal amounts of each fraction were subjected to Western blot analyses using anti-pSmad2 and anti-pSmad3 antibodies. The purity of the cytosolic and nuclear fractions was verified by the assessment of GAPDH and HDAC1, respectively. (B) 2ME2 enhances Smad3–β2-tubulin association and antagonizes TGF-β3–induced dissociation of this complex in huLM cells. Serum-starved huLM cells were pretreated with 2ME2 or vehicle for 1 hour and then exposed to TGF-β3 for 30 minutes. Whole-cell extracts were subjected to reciprocal coimmunoprecipitation analysis with anti-Smad3 (I) and anti–β2-tubulin (II) antibodies. After immunoblotting analysis, the blot was stripped and then probed with anti-Smad3 or anti–β2-tubulin, respectively (input). (C) Expression of total α-tubulin, detyrosinated α-tubulin, and acetylated α-tubulin in huLM cells treated with TGF-β3 and/or 2ME2. Abbreviations as in Figures 1 and 2. Fertility and Sterility 2012 98, 178-184.e1DOI: (10.1016/j.fertnstert.2012.04.002) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Effect of 2ME2 and/or TGF-β3 on PI3K/Akt/mTOR activation in huLM cells. Cell lysates were collected 48 hours after treatment and subjected to immunoblotting for pS6, S6, pAKT (Ser473), or pan-AKT. β-Actin was used as loading control. Fertility and Sterility 2012 98, 178-184.e1DOI: (10.1016/j.fertnstert.2012.04.002) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions