Development and validation of a three-dimensional in vitro model for uterine leiomyoma and patient-matched myometrium Minnie Malik, Ph.D., William H.

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Development and validation of a three-dimensional in vitro model for uterine leiomyoma and patient-matched myometrium Minnie Malik, Ph.D., William H. Catherino, M.D., Ph.D. Fertility and Sterility Volume 97, Issue 6, Pages 1287-1293 (June 2012) DOI: 10.1016/j.fertnstert.2012.02.037 Copyright © 2012 Terms and Conditions

Figure 1 Three-dimensional (3D) cultures stained for F-actin (phalloidin 488): (A) myometrial cells and (B) leiomyoma cells. Both cultures demonstrate the spindle-shaped characteristic of the cells. (C) Leiomyoma cells seen in two-dimensional (2D) culture and under ×20 magnification demonstrate a more flattened format and parallel strands of F-actin. (D) Leiomyoma cells in 3D cultures also stained positive for smooth muscle cell–specific α-actin. Fertility and Sterility 2012 97, 1287-1293DOI: (10.1016/j.fertnstert.2012.02.037) Copyright © 2012 Terms and Conditions

Figure 2 Cytoimmunoflourescence of three-dimensional (3D) leiomyoma cultures, demonstrating protein location and bioactivity. (A) Leiomyoma cultures stained with antibody to smooth muscle cell-α-actin protein, demonstrating a network of cells. (B) The 3D culture exposed to fibronectin antibody (green fluorescence), highlighting the protein in the matrix. (C) Loss of the network when 3D cultures are exposed to integrin-β1 function-inhibiting antibody at 1 μg/mL. (D) Red fluorescence indicative of cells exposed to caspase-3–specific antibody after an initial treatment of the 3D cultures with 1 μg/mL of integrin-β1 antibody. (E) As negative control, leiomyoma 3D cultures were exposed to primary antibody but not to labeled secondary antibody. (F) Cultures devoid of cells but undergoing same treatment as culture in A, indicating lack of autofluorescence of the collagen-1 matrix. DAPI-binding of the nuclei (blue fluorescence) is used in all cultures to specify the location of the leiomyoma cells. Fertility and Sterility 2012 97, 1287-1293DOI: (10.1016/j.fertnstert.2012.02.037) Copyright © 2012 Terms and Conditions

Figure 3 Bioactivity of myometrial three-dimensional (3D) cultures. The cultures were treated to either 0 ng/mL (control) or 10 ng/mL of transforming growth factor-β3 (TGF-β3) before RNA isolation and quantitative reverse-transcriptase polymerase chain reaction. The RNA transcripts were up-regulated for collagen-1, fibronectin, and versican, and down-regulated for dermatopontin. The expression of all genes was normalized against 18S as reference gene. Fertility and Sterility 2012 97, 1287-1293DOI: (10.1016/j.fertnstert.2012.02.037) Copyright © 2012 Terms and Conditions