Changes related to phosphatidylinositol 3-kinase/Akt signaling in leiomyomas: possible involvement of glycogen synthase kinase 3α and cyclin D2 in the pathophysiology  Laila Karra, M.Sc., Asher Shushan, M.D., Assaf Ben-Meir, M.D., Nathan Rojansky, M.D., Benjamin Y. Klein, M.D., David Shveiky, M.D., Rubina Levitzki, B.Sc., Hanna Ben-Bassat, Ph.D.  Fertility and Sterility  Volume 93, Issue 8, Pages 2646-2651 (May 2010) DOI: 10.1016/j.fertnstert.2009.03.100 Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Schematic representation of selected PI3K/Akt proteins controlling proliferation and/or apoptosis and their activation. The PI3K pathway, activated by many survival factors, leads to activation of PIP3, PDK1, and AKT, important players of survival signaling. PTEN negatively regulates the PI3K pathway. Active AKT inhibits by phosphorylation many downstream proapoptotic proteins: GSK3, FoxO1 (a member of the FKHR family), and Bax (a member of the Bcl2 family). Active AKT also is involved in suppressing the activity (by phosphorylation) of tuberin and hamartin—critical regulators for cell growth and proliferation. Akt regulates cell growth also through its effect on the cell cycle and on the level of D type cyclins (CD1/2). Regulation of D-type cyclins also involves GSK3. → Direct stimulatory modification; —| Direct inhibitory modification; P = phosphorylated. Colors indicate protein function: red = kinase; green = phosphatase; blue = transcription factor. Fertility and Sterility 2010 93, 2646-2651DOI: (10.1016/j.fertnstert.2009.03.100) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 2 (A) Representative immunoblot analysis (Western blot) of pGSK3, GSK3, cyclin D2, and β-actin of paired leiomyoma (L) and myometrium (M) cell cultures (n = 4). Cells were seeded at 7 to 9 × 105 cells/25 cm2 flasks in phenol red–free DMEM and 10% charcoal-stripped fetal bovine serum. After 48 hours the cell cultures were washed twice with 20 mL cold phosphate-buffered saline solution and analyzed. (B) Summary of the examined protein level of leiomyoma (Leio) and myometrial cell (Myo) cultures (n = 11) determined by densitometry measurements of the Western blot results and expressed in arbitrary units. Box plots indicate the upper and lower quartiles, with the horizontal line representing the median. The whiskers (above and below boxes) correspond to the highest and lowest value respectively that is not an outlier. Circles represent extreme values (Wilcoxon signed rank test analysis). Fertility and Sterility 2010 93, 2646-2651DOI: (10.1016/j.fertnstert.2009.03.100) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions