Figure 1. Representative schematic diagram summarizing the construction of a competitive PCR primer for uPA. An internal standard fragment was constructed by deletion of a 203-bp fragment from the specific target cDNA to be detected. Published in: Chun-Shan Chou; Hua Zhu; Eliezer Shalev; Colin D. MacCalman; Peter C. K. Leung; The Journal of Clinical Endocrinology & Metabolism 2002, 87, DOI: /jc Copyright © 2002

Figure 2. Standard curves generated for uPA and PAI. Photomicrograph of an ethidium bromide-stained gel containing uPA or PAI-1 specific PCR products generated by the coamplification of a fixed amount of target cDNA (1 μl) and serial dilutions of concentrations of competitive cDNA (8, 4, 2, 1, 0.5, 0.25, pg/μl) (A and B, upper panel). The two lines cross in the range of 0.5–1 pg/μl and 1 pg/μl internal standard cDNA added for uPA and PAI-1, indicating that approximated 1 pg uPA and PAI-1 cNDA could be detected after RT of 1 μg total RNA (A and B, lower panel). Increasing amounts of target cDNA were coamplified with 1 pg/μl competitive cDNA. The intensities of the ratio of target and competitive cDNA generated from these reaction mixtures were determined (C and D, upper panel). The log ratios of target competitive product density plotted against the log amount of target initially added to the PCR reactions are shown in the graphs below (C and D). Published in: Chun-Shan Chou; Hua Zhu; Eliezer Shalev; Colin D. MacCalman; Peter C. K. Leung; The Journal of Clinical Endocrinology & Metabolism 2002, 87, DOI: /jc Copyright © 2002

Figure 3. QC-PCR analysis of the effects of GnRH I or GnRH II on uPA and PAI mRNA levels in EVTs. Time-dependent effects on uPA (A and B) and PAI-1 (C and D) were determined by culturing the cells in the presence or absence of GnRH I (A and C) or GnRH II (B and D) for 0–48 h (lanes 1–6, respectively). Representative photomicrographs of the corresponding ethidium-stained gels are shown in the upper panels. A 100-bp DNA ladder is shown in lane M with the size of the target and competitive cDNAs indicated on the left. The gels were analyzed using UV densitometry. Data are presented as the means of five individual experiments and are presented as the mean ± se (a, P < vs. untreated control; b, P < 0.05 vs. untreated control) in the bar graphs shown in the lower panels. Published in: Chun-Shan Chou; Hua Zhu; Eliezer Shalev; Colin D. MacCalman; Peter C. K. Leung; The Journal of Clinical Endocrinology & Metabolism 2002, 87, DOI: /jc Copyright © 2002

Figure 4. Effects of GnRH I and GnRH II of uPA mRNA and protein expression levels in cultured EVTs. Dose-dependent effects were determined by culturing the cells in increasing concentrations (0–100 nm; lanes 1–5, respectively) of GnRH I (A) or GnRH II (B) for 24 h. Representative photomicrographs of the corresponding ethidium-stained gels are shown in the upper panels. A 100-bp DNA ladder is shown in lane M with the size of the expected PCR products indicated on the left. ELISA analysis of uPA expression level in conditioned medium of isolated EVTs cultured in the presence of an increasing concentration of GnRH I (C) or GnRH II (D). One milligram of protein from conditioned medium was used in each ELISA. Data are shown as the means of five individual experiments and are presented as the mean ± se (a, P < vs. untreated control) in the bar graphs shown in the lower panels. Published in: Chun-Shan Chou; Hua Zhu; Eliezer Shalev; Colin D. MacCalman; Peter C. K. Leung; The Journal of Clinical Endocrinology & Metabolism 2002, 87, DOI: /jc Copyright © 2002

Figure 5. Effects of GnRH I and GnRH II of PAI-1 mRNA and protein expression levels in cultured EVTs. QC-PCR analysis of PAI-1 mRNA levels were determined by culturing the cells in increasing concentrations (0–100 nm; lanes 1–5, respectively) of GnRH I (A) or GnRH II (B) for 24 h. Representative photomicrographs of the corresponding ethidium-stained gels are shown in the upper panels. A 100-bp DNA ladder is shown in lane M with the size of the expected PCR products indicated on the left. The blots were analyzed using UV densitometry. ELISA analysis of PAI-1 expression level in conditioned medium of isolated EVTs cultured in the presence of an increasing concentration of GnRH I (C) or GnRH II (D). One milligram of protein from conditioned medium was used in each ELISA. Data are shown as the means of five individual experiments and are presented as the mean ± se (a, P < vs. untreated control. b, P < 0.05 vs. untreated control) in the bar graphs shown in the lower panels. Published in: Chun-Shan Chou; Hua Zhu; Eliezer Shalev; Colin D. MacCalman; Peter C. K. Leung; The Journal of Clinical Endocrinology & Metabolism 2002, 87, DOI: /jc Copyright © 2002

Figure 6. Effects of Cetrorelix on the GnRH I- or GnRH II-mediated regulation of EVT uPA mRNA and protein expression levels. QC-PCR analysis of the EVTs. Cells were cultured in the presence of 100 nm GnRH I (A) or GnRH II (B) and an increasing amount of Cetrorelix. Representative photomicrographs of the corresponding ethidium stained gels are shown in the upper panels. A 100-bp DNA ladder is shown in lane M with the size of the target and competitive cDNAs indicated on the left hand side. The blots were analyzed using UV densitometry. ELISA analysis of uPA expression level in conditioned medium of isolated EVT cultured in the presence of an increasing concentration of GnRH I (C) or GnRH II (D). One milligram of protein from the conditioned medium was loaded in each ELISA reaction. Data are shown as the means of five individual experiments, and are presented as the mean ± se (a, P < vs. 100 nm GnRH, b, P < 0.05 vs. 100 nm GnRH) in the bar graphs shown in the lower panels. Published in: Chun-Shan Chou; Hua Zhu; Eliezer Shalev; Colin D. MacCalman; Peter C. K. Leung; The Journal of Clinical Endocrinology & Metabolism 2002, 87, DOI: /jc Copyright © 2002

Figure 7. Effects of Cetrorelix on the GnRH I- or GnRH II-mediated regulation of EVT PAI-1 mRNA and protein expression levels. QC-PCR analysis of EVTs. Cells were cultured in the presence of 100 nm GnRH I (A) or GnRH II (B) and an increasing amount of Cetrorelix. Representative photomicrographs of the corresponding ethidium stained gels are shown in the upper panels. A 100-bp DNA ladder is shown in lane M with the size of the target and competitive cDNAs indicated on the left. The blots were analyzed using UV densitometry. ELISA analysis of PAI-1 expression level in conditioned medium of isolated EVT cultured in the presence of GnRH I (C) or GnRH II (D) and an increasing amount of Cetrorelix. One milligram of protein from the conditioned medium was loaded in each ELISA reaction. Data are shown as the means of five individual experiments, and are presented as the mean ± se (a, P < vs. 100 nm GnRH, b, P < 0.05 vs.100 nm GnRH) in the bar graphs shown in the lower panels. Published in: Chun-Shan Chou; Hua Zhu; Eliezer Shalev; Colin D. MacCalman; Peter C. K. Leung; The Journal of Clinical Endocrinology & Metabolism 2002, 87, DOI: /jc Copyright © 2002

Figure 8. Line graph depicting the ratio of uPA/PAI-1 expression levels in conditioned medium of EVTs cultured in the presence of increasing concentrations of GnRH I or GnRH II (0–100 nm). Published in: Chun-Shan Chou; Hua Zhu; Eliezer Shalev; Colin D. MacCalman; Peter C. K. Leung; The Journal of Clinical Endocrinology & Metabolism 2002, 87, DOI: /jc Copyright © 2002