Role of interleukin-1 receptor type II in the pathogenesis of endometriosis Zhen Hou, Ph.D., Jing Zhou, M.Sc., Xiang Ma, Ph.D., Lu Fan, M.Sc., Lianming.

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Role of interleukin-1 receptor type II in the pathogenesis of endometriosis Zhen Hou, Ph.D., Jing Zhou, M.Sc., Xiang Ma, Ph.D., Lu Fan, M.Sc., Lianming Liao, Ph.D., Jiayin Liu, Ph.D., M.D. Fertility and Sterility Volume 89, Issue 1, Pages 42-51 (January 2008) DOI: 10.1016/j.fertnstert.2007.01.044 Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Restriction enzyme digestion analysis of homologous recombinant adenovirus plasmid by PacI. After cloning the ORF of human IL-1RII gene (approximately 1200 bp) and homologous recombination with an adenoviral backbone, the positive clones were amplified by PCR and verified by PacI restriction analysis and sequencing. Arrow: 4.5 kb or 3.0 kb band cut by PacI. M: 15 kb Marker. 1: Uncut pAd-RII. 2: Positive control. 3: pAd-RII PacI number 1. 4: pAd-RII PacI number 2. 5: pAd-LacZ PacI. Fertility and Sterility 2008 89, 42-51DOI: (10.1016/j.fertnstert.2007.01.044) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Analysis of the PCR product of Ad-RII by agarose gel electrophoresis. After homologous recombination into an adenoviral backbone, the size of the rAd-RII construct was confirmed by whole genome PCR. Arrow: 1.2 kb band. M: 2.0 kb Marker. 1: Positive control. 2: Ad-RII number 1. 3: Ad-RII number 2. 4: Ad-LacZ number 1. 5: Ad-LacZ number 2. Fertility and Sterility 2008 89, 42-51DOI: (10.1016/j.fertnstert.2007.01.044) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Detection of IL-1RII and LacZ expression in infected ESCs by immunohistochemical analysis and X-Gal staining (×100). (A) ESCs infected with AD-RII (MOI = 1000). (B) Negative control. (C) ESCs infected with rAd-LacZ (MOI = 1000). (D) Negative control. Fertility and Sterility 2008 89, 42-51DOI: (10.1016/j.fertnstert.2007.01.044) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Effect of sIL-1RII on IL-6/8 protein secretion stimulated by IL-1β. Duplicate cultures from 4 independent experiments. (A) IL-8 secretion was decreased by 34.43% ± 8.08%. (B) IL-6 secretion was decreased by 38.32% ± 5.71%. ∗: P<.05. (=): Negative control (untreated ESCs). PBS: ESCs treated with IL-1β. sRII: ESCs treated with IL-1β and sIL-1RII. Fertility and Sterility 2008 89, 42-51DOI: (10.1016/j.fertnstert.2007.01.044) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 5 Effect of rAd-RII on IL-6/8 expression after IL-1β stimulation. These experiments were performed in duplicate for three independent experiments. (A) Infection of rAd-RII decreased IL-8 expression by 36.34% ±11.2%. ∗: P<.05, rAd-RII vs. PBS or rAd-LacZ. ∗∗: P>.05 rAd-LacZ vs. PBS). (B) Secretion of IL-6 increased after infection of rAd-RII or rAd-LacZ, compared with PBS group, while compared with rAd-LacZ group IL-6 was not inhibited significantly by rAd-RII P>.05. (=): Negative control (untreated ESCs). PBS: ESCs treated with IL-1β. Ad-LacZ: ESCs infected with Ad-LacZ and stimulated with IL-1β. Ad-RII: ESCs infected with AD-RII and stimulated with IL-1β. Fertility and Sterility 2008 89, 42-51DOI: (10.1016/j.fertnstert.2007.01.044) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 6 Representative image of silver-stained 2-D reference maps of ESCs stimulated by IL-β (1.0 ng/mL). Proteins extracted from treated endometrial cells were separated in 2-DE gels and 2,800–3,000 spots on each gel (silver-stained) were detected. (A) PBS control group. (B) Infection of rAd-RII group. Number labels: The 62 spots that showed a significant difference between the rAd-RII group and the PBS control group. Fertility and Sterility 2008 89, 42-51DOI: (10.1016/j.fertnstert.2007.01.044) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 7 Location and peptide masses of two sample spots. The location of protein GBLP and hnRNPA1 detected by the ImageMaster 2D Platinum software can be seen from the picture selected from the 6 gels that were matched. Peptide masses of the two proteins were obtained by MALDI-TOF mass spectrometry, and the mass spectra interpretations were screened against the automatic protein identification tool. (2574) Guanine nucleotide-binding protein (GBLP). (7077) Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1). Fertility and Sterility 2008 89, 42-51DOI: (10.1016/j.fertnstert.2007.01.044) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions