1. The WNT/β-catenin signaling pathway and expression of survival promoting genes in luteinized granulosa cells: endometriosis as a paradigm for a dysregulated apoptosis pathway 
Ana M. Sanchez, Ph.D., Paola Viganò, Ph.D., Federica Quattrone, D.Sc., Luca Pagliardini, Ph.D., Enrico Papaleo, M.D., Massimo Candiani, M.D., Paola Panina-Bordignon, Ph.D. 
Fertility and Sterility 
Volume 101, Issue 6, Pages (June 2014)
DOI: /j.fertnstert
Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

2. Figure 1
Expression of non-canonical WNT pathway members (A) WNT4, (B) WNT5a, (C) sFRP4, and (D) sFRP5 and canonical WNT pathway members (E) WNT1 and (F) WNT3 mRNA in luteinized granulosa cells as evaluated by real-time quantitative polymerase chain reaction analysis. Cells were obtained from 33 women who underwent intracytoplasmic sperm injection because of male infertility (control) and 30 women with a diagnosis of endometriosis. Total RNA isolated from the cells was quantified with the use of the 18S gene as an endogenous control. Data were analyzed with the use of the comparative Ct method and are expressed as mean and SD of relative expression values corresponding to 2−ΔCt. ∗P<.05 compared with the control group.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

3. Figure 2
(A) Nonphosphorylated β-catenin (Ser33/37/Thr41) active form, total β-catenin protein levels, and Ser9-phosphorylated glycogen synthase kinase (GSK) 3β in luteinized granulosa cells were assayed by western blotting. (B) Data were expressed as mean and SD of relative density derived from the quantitative analysis of the blot images, referring to seven samples from women who underwent intracytoplasmic sperm injection because of male infertility (control) and seven samples from women with a diagnosis of endometriosis. ∗P<.05 and ∗∗P<.01 compared with the control group. (C) β-Catenin location in luteinized granulosa cells was assessed by immunofluorescence assay with anti–β-catenin antibody. β-Catenin location (green) and 4′,6-diamidino-2-phenylindole (DAPI)–stained nuclei (blue) are shown.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

4. Figure 3
Expression of (A) survivin and (B) BMP4 mRNA in luteinized granulosa cells as evaluated by real-time quantitative polymerase chain reaction analysis. Cells were obtained from 33 women who underwent intracytoplasmic sperm injection because of male infertility (control) and 30 women with a diagnosis of endometriosis. Total RNA isolated from the cells were quantified with the use of the 18S gene as an endogenous control. Data were analyzed with the use of the comparative Ct method and are expressed as mean and SD of relative expression corresponding to 2−ΔCt. ∗P<.05; ∗∗P<.01; compared with the control group.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

5. Figure 4
Analysis of DNA histograms of luteinized granulosa cells by flow cytometry. (A) Different regions correspondent to cells undergoing apoptosis (subdiploid levels of DNA) and to the G1, S, and G2/M phases of the cell cycle. Cells were obtained from four women who underwent intracytoplasmic sperm injection because of male infertility (control) and four women with a diagnosis of endometriosis. Data are expressed as mean percentage of cells and SD in the various regions. ∗P<.05 compared with the control group. (B) A pool of luteinized granulosa cells from four healthy control patients were plated and cultured in complete medium for 24 hours. Cells were then treated with rhWNT4, rhWNT5, or both at 200 ng/mL for 48 hours, and cell viability was determined by dimethyl thiazolyl diphenyl tetrazolium assay. Results are presented as the mean and SD of three independent experiments performed in triplicate relative to vehicle-treated cells. ∗P<.05 compared with the untreated group. Total β-catenin protein levels in luteinized granulosa cells under these conditions were assayed by Western blotting.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions