1. The growth hormone–releasing hormone (GHRH) antagonist JV-1-36 inhibits proliferation and survival of human ectopic endometriotic stromal cells (ESCs) and the T HESC cell line 
Marta Annunziata, Ph.D., Cristina Grande, Francesca Scarlatti, Ph.D., Francesco Deltetto, M.D., Elena Delpiano, M.D., Marco Camanni, M.D., Ezio Ghigo, M.D., Riccarda Granata, Ph.D. 
Fertility and Sterility 
Volume 94, Issue 3, Pages (August 2010)
DOI: /j.fertnstert
Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

2. Figure 1
Splice variant (SV)1 and growth hormone–releasing hormone receptor (GHRH-R) mRNA expression in eutopic and ectopic endometriotic tissues and primary endometriotic stromal cells (ESCs), evaluated by standard (SV1 and β-actin) or nested RT-PCR (GHRH-R). Lane 1: negative control; lane 2: positive control (human pituitary); lanes 3–5: eutopic endometriotic tissues; lanes 6–8: ectopic endometriotic tissues; lanes 9–10: ESCs from ectopic endometriotic tissues. As expected, the amplified products corresponded to 523 bp for SV-1, 191 bp for GHRH-R, and 230 bp for β-actin, which was used as control gene.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

3. Figure 2
Effect of the GHRH antagonist JV-1-36 on ectopic endometrial stromal cell (ESC) proliferation, assessed by BrdU incorporation. The ESCs were incubated for 48 hours in serum-free medium (SF), or in the presence of serum, either with or without (c = control) the indicated concentrations of JV Data, expressed as percent of control, are the mean ± SE of three independent experiments from three different tissues. Each experiment was performed in quintuplicate. ∗P<.05; ∗ ∗P<.01; vs. control.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

4. Figure 3
Effect of the growth hormone–releasing hormone antagonist JV-1-36 on ectopic endometrial stromal cell (ESC) survival. The ESCs were serum starved for 12 hours and incubated for 48 hours in serum-free (SF) medium (c = control), in the presence of the indicated concentrations of JV (A) ESC survival assessed by MTT. The results, expressed as percent of control, are the mean ± SE of five independent experiments, each performed in quintuplicate, using cells from different tissues. ∗P<.05; ∗ ∗P<.01; vs. control). (B) ESC cell death assessed by Trypan blue exclusion. Values are the mean ± SE of three independent experiments, using cells from different tissues. ∗P<.05 vs. control.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

5. Figure 4
Growth hormone–releasing hormone (GHRH), splice variant (SV)1, and GHRH receptor (GHRHR) mRNA expression in the T HESC human endometrial stromal cell line and inhibitory effect of JV-1-36 on T HESC survival. (A) GHRH, SV-1, and GHRH R mRNA evaluated by standard (SV-1 and β-actin) and nested (GHRH and GHRH R) reverse-transcription polymerase chain reaction. L: 100-bp ladder; c(−): negative control. (B) Effect of JV-1-36 on T HESC survival, assessed by MTT. Cells were starved for 24 hours and then incubated for 72 hours in serum-free medium (c = control) in the presence of JV-1-36 at the indicated concentrations. Data are expressed as percent of control and are the mean ± SE of four independent experiments, each performed in quadruplicate. ∗P<.05; ∗∗P<.01; vs. control.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

6. Figure 5
Effect of JV-1-36 on cyclic adenosine monophosphate (cAMP) production, ERK1/2 phosphorylation, and insulin-like growth factor (IGF)-2 mRNA expression in the T HESC human endometrial stromal cell line. (A) Cells were treated for up to 30 minutes with JV-1-36 (1 or 10 μmol/L), in the presence of 100 μmol/L isobutylmethylxanthine. Data are expressed as percent of basal levels (c) and are the mean ± SE of three independent experiments performed in duplicate. ∗P<.05; ∗∗P<.01 vs. control). (B) Western blot analysis on whole cell lysates obtained after 72 hours' treatment with JV-1-36 (10 μmol/L) in serum-free medium. Equal protein loading was determined by reprobing the membrane with antibody to total ERK1/2. Results are representative of three independent experiments. (C) T HESC survival, evaluated by MTT in cells cultured in the presence of serum or in serum-starved medium for 72 hours (after 24 hours' starvation) either with or without MDL (25 nmol/L) and PD98059 (40 μmol/L). Data are expressed as percent of control (c, serum-free medium) and are the mean ± SE of three independent experiments performed in quintuplicate. ∗P<.05; ∗∗P<.01; vs. control). (D) Effect of 4 hours' or 24 hours' treatment with JV-1-36 (10 μmol/L) on IGF-2 mRNA level in T HESCs, assessed by real-time polymerase chain reaction. Results, normalized to GAPDH transcript, are expressed as fold over untreated cells and are the mean ± SE of three separate experiments. ns = not significant.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions