Expression of insulin-like growth factors (IGFs) and IGF signaling: molecular complexity in uterine leiomyomas  Lan Peng, M.D., Yong Wen, M.D., Yulong Han, M.D., Anran Wei, Guizhi Shi, M.D., Masashi Mizuguchi, M.D., Peng Lee, M.D., Eva Hernando, Ph.D., Khush Mittal, M.D., Jian-Jun Wei, M.D.  Fertility and Sterility  Volume 91, Issue 6, Pages 2664-2675 (June 2009) DOI: 10.1016/j.fertnstert.2007.10.083 Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Differential expression of IGF-1 mRNA and protein in fibroids. (A) Semiquantitative RT-PCR analysis of IGF-1 cDNA in fibroids (even numbers) and matched myometrium (odd numbers). G3PDH was used as an internal control. (B) Arrow lines illustrating the normalized values of IGF-1/G3PDH cDNA (y axis, %) in fibroids and matched myometrium. No statistical significance of mean values between fibroids and myometrium was noted. (C) Dot plot of the relative amounts of IGF-1 cDNA in the secretory (SE) and proliferative (PE) phases. Each dot represents an individual case. (D) Significant differences of mean values of IGF-1 expression between SE and PE in both myometrium and fibroids. (E) Photomicrographs of immunoreactivities for IGF-1 in fibroids and matched myometrium. (F) Significant analysis of the net gains of IGF-1 immunoreactivity associated with tumor sizes. ∗P<.05; ∗∗P<.01. The tumor sizes and numbers of cases are listed below the histogram bars. y axis in B, C, and D indicates percentage of IGF-1/G3PDH ratio. Fertility and Sterility 2009 91, 2664-2675DOI: (10.1016/j.fertnstert.2007.10.083) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Differential expression of IGF-2 and different usages of IGF-2 promoters in fibroids. (A) Photonegative images of the multiplex RT-PCR products representing the cDNA from specific IGF-2 isoforms in large (L-ULM) and small (S-ULM) fibroids, and matched myometrium (control) in eight cases. Primers used for the RT-PCR are listed on the right (see B). G3PDH was used as a RNA loading control. (B) A sketch diagram illustrating genomic organization of IGF-2, including genomic DNA (double line) and relative locations of each of 10 coding (black box) and non-coding (open box) exons. Promoters P1 to P4 in exons 1, 4, 6, and 7 are shown on top. The forward (F) and reverse (R) primers for the amplification of specific isoforms from different promoter usage are shown below. (C) The relative abundances of IGF-2 cDNA products (IGF-2/G3PDH) and the significance values from each isoform were analyzed and are listed in the histogram. (D) Photomicrographs illustrating an example of immunoreactivity for IGF-2 in fibroids and matched myometrium (∗P<.05). (E) Mean values of net gain of IGF-2 gene products in different tumor sizes. There was a positive correlation between IGF-2 immunoreactivity and tumor size, but no statistical significance was obtained (P>.05). The tumor sizes and numbers of cases are listed below. Fertility and Sterility 2009 91, 2664-2675DOI: (10.1016/j.fertnstert.2007.10.083) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Methylation analysis of IGF-2 promoter 1 and 2 in fibroids and myometrium. (A) Two cases with significant over-expression of IGF-2 in both large (L) and small (S) fibroids (using IGF-2 P1 and P2) were selected for methylation analysis. (B) Sketch diagram of the genomic DNA immediately upstream of IGF-2 P1 (518 base pairs) and P2 (485 base pairs) selected for methylation analysis. The sites of restriction enzyme SmaI and HaeIII were indicated by arrows. Arrow of SmaI∗ is a polymorphic site. (C) Methylation analysis of IGF-2 P1 and P2 in fibroids and matched myometrium (MM). Insulin-like growth factor-2 promoter DNAs with and without bismuth treatment were amplified by PCR, then digested with SmaI (P1) and HaeIII (P2), and size-fractionated in agarose gels. The methylation patterns of P1 and P2 in fibroids were identical to that in myometrium, in which only one allele had high levels of methylation. Fertility and Sterility 2009 91, 2664-2675DOI: (10.1016/j.fertnstert.2007.10.083) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Differential expressions of p-AKT and p-S6K in large cohort fibroids and matched myometrium. (A) Photomicrographs illustrating examples of distinct and moderate immunoreactivities for p-AKT and p-S6K in fibroids, but not in myometrium. (B) The percentage of p-AKT- and p-S6K-positive fibroids in a total of 180 cases. (C, D) Differential expressions of p-AKT (C) and p-S6K (D) in association with tumor size. The net gain values of immunoreactivity from fibroids against myometrium (ULM-MM) are shown on the y axis. The tumor sizes and case numbers are listed below the histogram bars. (E) Example of increased p-AKT, but not total AKT protein, in fibroids (ULM) in comparison to matched myometrium (MM). Actin was used as loading control. Fertility and Sterility 2009 91, 2664-2675DOI: (10.1016/j.fertnstert.2007.10.083) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 5 Differential expressions of TSC gene products hamartin and tuberin in fibroids. (A) Photomicrographs illustrating the patterns of immunoreactivity for hamartin and tuberin in fibroids and myometrium. (B) Differential expression of hamartin and tuberin in association with tumor size. The net gain values of immunoreactivity from fibroids against myometrium (ULM-MM) are shown on the y axis. The tumor sizes and case numbers are listed below the histogram bars. (C) Differential expression of hamartin and tuberin in five small (S) and five large (L) fibroids by Western blot analysis. Four matched myometrium (MM) were used as controls. Erk was used as a protein loading control. (D) Quantitative analysis of hamartin and tuberin protein product in fibroids (ULM) and myometrium (MM) scaled from Western blot bands listed in C and normalized by Erk protein (y axis). There was significant over-expression of hamartin and under-expression of tuberin in fibroids compared with matched myometrium (P<.05). No significance was found between large and small fibroids. Fertility and Sterility 2009 91, 2664-2675DOI: (10.1016/j.fertnstert.2007.10.083) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions