The data are shown in a dose-dependent Cytoskeletal Signaling inhibitor manner. Figure 3 Effects of recombinant human Mullerian-inhibiting substance (MIS)/anti-Mullerian hormone (E.Coli derived) on endometriosis stromal cell line. (A) pre-G1 fraction analysis of endometriosis stromal cells treated for 24-48-72 hrs with the indicated final concentrations of MIS. The data are shown in a time-dependent manner. (B) pre-G1 fraction analysis of endometriosis stromal cell line treated for 24-48-72 hrs with the indicated final concentrations of MIS. The data are shown in a dose-dependent manner. (C) Cell
cycle analysis of endometriosis stromal cells treated for 24-48-72 hrs with the indicated final concentrations of MIS. The data are shown in a time-dependent manner. (D) Cell cycle analysis of endometriosis stromal cells treated for 24-48-72 hrs with the indicated final concentrations
of MIS. The data are shown in a dose-dependent manner. Figure 4 Effects of purified recombinant protein of Homo Sapiens anti-Mullerian hormone (AMH) on endometriosis stromal cell line. (A) Cell cycle analysis of endometriosis stromal cells treated for 48 hrs with AMH at 1000 ng/mL. Proteasome inhibition assay (B) pre-G1 fraction analysis of endometriosis stromal cells treated for 48 hrs with AMH at 1000 ng/mL. Figure 5 Analysis of AMH, AMHRII expression and CytP450 activity. (A) Real-time PCR to assess the percentage of expression levels of AMH (1), AMH (2), AMH type II Receptor (1) and (2) (AMH RII) genes in endometrial epithelial and stromal cell line respectively. (B) Expression levels of the Cytochrome P4501 and 2 isoforms and Reverse Transcriptase–Polymerase Chain Reaction (RT-PCR) for the CytP (450) 1 and 2 in epithelial and stromal cell line respectively; GAPDH represents loading control. (C) CYP Activity assay in endometrial stromal cells treated for 24 hrs at 1000 ng/mL of MIS
full-length. (D) CYP Amylase Activity assay in endometrial stromal cells treated for 24 hrs at 1000 ng/mL of Plasmin-cleaved MIS. Considering that the plasmin-digested AMH has been reported to be more active in cultured human endometrial cell lines [15], human plasmin was used to cleave and activate the recombinant Human AMH at its monobasic arginine-serine site at residues 427-428 and then tested in functional experiments on both endometriosis stromal and epithelial cells. Firstly, we found that plasmin-digested AMH can alter the expression or function of CYP19, evaluated by testing CYP19 activity. The results suggest that the plasmin-digested AMH was able to suppress most of the CYP19 activity. When the plasmin-digested AMH was used on both endometriosis stromal and epithelial cells (Figure 6), an increase of pre-G1 phase treating with plasmin-digested AMH in both cell lines was detected, most marked in the epithelial cells (Figure 6). Also the effect on induction of apoptosis was stronger during the first 24 hours of treatment (Figure 6A-B).