Gene expression profiling of multiple leiomyomata uteri and matched normal tissue from a single patient  Irina K. Dimitrova, M.D., Jennifer K. Richer, Ph.D., Michael C. Rudolph, B.S., Nicole S. Spoelstra, B.S., Elaine M. Reno, B.S., Theresa M. Medina, B.S., Andrew P. Bradford, Ph.D.  Fertility and Sterility  Volume 91, Issue 6, Pages 2650-2663 (June 2009) DOI: 10.1016/j.fertnstert.2008.03.071 Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 1 (A) The statistically significant differentially expressed genes that exhibited consistent changes in all three tumors relative to normal myometrium of a magnitude of at least twofold. (B) Heat map showing cluster analysis of the differentially expressed 816 genes in three uterine leiomyoma and three samples of matched adjacent normal myometrium. From left to right, the first three columns represent the three tumors followed by the corresponding normal tissue. Rows represent individual genes. Red, increased gene expression; green, decreased gene expression. The color intensity is proportional to the hybridization intensity of a gene from its median level across all samples. Fertility and Sterility 2009 91, 2650-2663DOI: (10.1016/j.fertnstert.2008.03.071) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Semiquantitative RT-PCR analysis. RNA from paired myometrium/leiomyoma from five different patients was analyzed using specific primer pairs, as indicated and described in Materials and Methods and electrophoresed on agarose gels. Bands were quantitated by scanning densitometry and expressed as relative intensity normalized to GAPDH. B2, B3, B4, B9, B11— patient designations. N1–5—normal myometrium; L1–5—corresponding leiomyoma samples. Fertility and Sterility 2009 91, 2650-2663DOI: (10.1016/j.fertnstert.2008.03.071) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 3 (A) Semiquantitative RT-PCR analysis of MMP11 (Stromelysin 3) expression. RNA from paired myometrium/leiomyoma from five different patients was analyzed using specific primer pairs, as indicated and described in Materials and Methods and electrophoresed on agarose gels. Bands were quantitated by scanning densitometry and expressed as relative intensity normalized to GAPDH. B2, B3, B4, B9, B11—patient designations. N1–5—normal myometrium; L1–5— corresponding leiomyoma samples. (B) Immunohistochemical staining for matrix metalloproteinase 11 (MMP-11) in normal myometrial tissue (top panels) and leiomyomata (bottom panels). Paraffin embedded sections were stained with anti-MMP-11 antibody as described in Materials and Methods. Representative 20× fields are depicted. B2, B3, B4, B9, B11—patient designations. N—normal myometrium, L—matched leiomyoma. Fertility and Sterility 2009 91, 2650-2663DOI: (10.1016/j.fertnstert.2008.03.071) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Western blot analysis of leiomyoma and myometrium samples from five patients using antibodies to (A) PKC β1, and (B) MST-4. Blots were reprobed for GAPDH as a loading control. Chemiluminescence was quantitated using a BioRad Chemdoc XRS imager and relative levels expressed as a ratio of leiomyoma to normal myometrium, normalized to GAPDH. B2, B4, B5, B8, and B9; patient designations, numbers indicate independent tumor/normal paired samples derived from that patient. N—myometrium, L—leiomyoma. Fertility and Sterility 2009 91, 2650-2663DOI: (10.1016/j.fertnstert.2008.03.071) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions