Fas ligand+ fallopian tube epithelium induces apoptosis in both Fas receptor+ T lymphocytes and endometrial cells  Sebastian E. Illanes, M.D., Kevin Maisey, M.Sc., Marcelo Sandoval, Ph.D., Felipe E. Reyes, Ph.D., Claudio Figueroa-Gaete, M.Sc., Alejandra Pérez-Sepúlveda, Ph.D., Maritza Busquets, M.D., Patricia González, M.Sc., Mónica Imarai, Ph.D.  Fertility and Sterility  Volume 100, Issue 2, Pages 550-560.e3 (August 2013) DOI: 10.1016/j.fertnstert.2013.04.013 Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Immunolocalization of FasL in the fallopian tube (FT). (A–D) FasL immunostaining of one representative sample of FT. Slices were sequentially incubated with the anti-FasL antibody and FITC-conjugated anti-goat IgG (green label). Tissue was counterstained with propidium iodide (red label). (B–D) FT images at ×40 magnification. Arrows indicate FasL labeling. L = lumen; S = stroma; E = epithelium; V = villous trophoblast. (E) Negative control. (F) Human placenta, positive control of FasL staining. (G) Western blot analysis of FasL from whole protein extracts. FasL− FT tissue (lane 1), FasL+ FT tissue (lane 2), HeLa cells (lane 3), Jurkat cells (lane 4). The arrow shows an immunoreactive protein of 37 kd. (H) FasL transcripts in human FT epithelial cells assessed by RT-PCR. FasL transcripts (upper panel), β-actin (bottom panel). PHA-activated Jurkat cells (lane 1), samples of human FT from eight different donors (lanes 2–9). FasL and β-actin PCR products were 406 and 838 bp, respectively. Products were separated by agarose gel electrophoresis and stained with ethidium bromide. Fertility and Sterility 2013 100, 550-560.e3DOI: (10.1016/j.fertnstert.2013.04.013) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Percentage of apoptotic T lymphocytes after incubation with FT epithelial cells. Apoptotic PHA-activated T lymphocytes were analyzed after a 5-hour coincubation with FT epithelial cells. Activated T cells cultured without FT cells were the negative control. The percentage of apoptotic cells was determined using a flow cytometry-coupled TUNEL assay. (A) Representative flow cytometry histogram of the percentage of TUNEL-positive T cells (right panel). The control is shown in the left panel. (B) Average of apoptotic T cells obtained from four different FT samples, each tested against peripheral blood cells isolated from three different donors. Bars represent the average ± standard error of the mean. *P<.05. Fertility and Sterility 2013 100, 550-560.e3DOI: (10.1016/j.fertnstert.2013.04.013) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Apoptosis of human endometrial cells induced after incubation with fallopian tube (FT) epithelial cells. Apoptosis was detected by TUNEL staining followed by confocal microscopy. The images shown in the left and center columns were taken in the red (propidium iodide staining) and green (TUNEL labeling) channels, respectively; overlay of images are shown in the right columns (yellow). Analysis of endometrial cells was done after 5 hours of culture. (A) Endometrial cells, negative control. (B) DNase I treatment, positive control. (C) Endometrial cells of healthy donors coincubated with FT epithelial cells. The images are representative of four independent experiments (four different donors). (D) Endometrial cells derived from a patient with endometriosis coincubated with FT epithelial cells. The micrographs were taken using a ×40 objective. The scale bars represent 50 μm. (E) The caspase-3 activity in endometrial cells induced after incubation with FT epithelial cells. The enzymatic activity of caspase-3 was assayed in lysates of endometrial cells treated with actinomycin D (apoptosis inducer) or with FT epithelial cells (FT Epi). The caspase-3 inhibitor Ac-DEVD-CHO (Inh) was used to test specificity. Data represent the mean ± standard error of the mean of four samples of isolated endometrial cells from normal donors. *Statistically significant difference from control (nontreated cultured endometrial cells). Fertility and Sterility 2013 100, 550-560.e3DOI: (10.1016/j.fertnstert.2013.04.013) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplementary Figure 1 Immunofluorescence analysis of primary cultures of fallopian tube (FT) epithelial cells. Primary cultures of FT epithelial cells were incubated with antibodies to cytokeratin-18 (A), vimentin (C), and FasL (E) followed by incubation with the corresponding FITC-labeled secondary antibody (green label). (B, D, F) Corresponding negative controls. Propidium iodide was used to counterstain the nucleus (red label). Fertility and Sterility 2013 100, 550-560.e3DOI: (10.1016/j.fertnstert.2013.04.013) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplementary Figure 2 Immunofluorescence analysis of FasR in human endometrial cells. (A) Ishikawa cells. (B) Negative control. (C) Endometrial cells END777. (D) Endometrial cells END 778. Cells were incubated with antibodies to FasR, followed by incubation with the corresponding FITC-labeled secondary antibody (green label). Propidium iodide was used to counterstain the nucleus (red label). Fertility and Sterility 2013 100, 550-560.e3DOI: (10.1016/j.fertnstert.2013.04.013) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions