Spheroids differentiation assay.

<p>20 day old spheroids derived from oviducts of 12 week chased mice were pulsed for one day with doxycycline and were subsequently allowed to differentiated for 0 (A), 1 (B and C), 2 (D and E), 3 (F) or 6 days (G) in the presence of 10% FCS. The two columns of images on the left hand side of the figure represent confocal images taken at two different planes (bottom and middle). The phase-contrast images in the third colum from the left represents a detail from the confocal image. C represents a spheroid derived from a different animal which was induced to differentiate for 1 day, displaying formation of multiple cell layers as indicated by nuclear DAPI staining and immunofluorescence for CK8. In E attachment of the spheroid to the culture dish is shown. H and I represent spheroids isolated from different animals that were allowed to differentiate for 9 days (H) and for 20 days (I) showing complex structures (nuclei were stained with Hoechst 34580).</p

Differentiation of spheroids towards specific cell lineages of the female reproductive tract.

<p>Early spheroids (2 days of culture, A), late spheroids (2 weeks of culture, B) and spheroids which were stimulated to differentiate (10% FCS added to the culture medium for 2 days, C) were stained for ERα, CD44, PR and PAEP expression and compared to stained sections from mice distal oviduct (D), proximal oviduct (E) and endometrium (F). The red arrows indicate ERα positive cells, the black arrows indicate ERα negative cells. In panel A, 4 different spheroids were used, in panel B two (ER and PR, CD44 and PEAP stainings were performed on the same spheroid, respectively) and stainings in panel C are on consecutive sections from one spheroid. Images are representative for three different experiments containing more than 10 early and late spheroids and 2 – 3 differentiated spheroids.</p

Characterization of identified LT-LRCs

<p>(<b>12 week chase</b>) <b>in mouse distal oviduct.</b> Immunofluorescent double-labeling was performed for GFP and ERα (A), GFP and PR (B), GFP and Ki67 (C), GFP and CD44 (D) and CD44 and ERα (E). DAPI staining was performed to display all cell nuclei. White arrowheads indicate Ki67 positive cells, the yellow arrowhead indicates a cell which is positive for CD44 as well as GFP.</p

Pulse-chase experiment using the doxycycline-inducible H2B-GFP system.

<p>A schematic representation of the experiment is given in A, where a 7-day treatment with doxycycline (pulse) results in expression of H2B-GFP throughout the entire organism. At different chase time points (12 and 47 weeks after pulse), the GFP signal is progressively diluted from dividing cells and retained in quiescent cells in the distal oviduct (B). In the proximal oviduct (C) and the endometrium (D), label-retaining cells (LRCs) are lost at both 12 and 47 weeks chase time points. FACSorting was performed on single cell digestions of oviducts from different mice. In (E), the GFP signal is plotted against the number of cells. Animals used were: untreated mice as negative controls (black line, Negative); mice pulsed for 7 days (no chase) as positive controls (red line, Pulse); mice pulsed for 7 days and chased for 12 weeks (green line, 12 weeks); and mice pulsed for 7 days and chased for 47 weeks (light-green line, 47 weeks). The scale bar represents 50 μM.</p

Isolated LT-LRC can form self-renewing spheroids in culture.

<p>Oviducts from 12 week chased mice were dissected and a single cell suspension was analyzed by FACS. From three different 12 week chased mice, GFP⁺ and GFP-negative (GFP−) cells were isolated by FACSorting and cultured in Matrigel under serum-free conditions to assay their capacity to form spheroids (A). Self-renewal was assayed by dissociating spheroids and plating the unsorted cell suspension to form new spheroids as indicated in the Materials and Methods section. In short, primary spheroids (i.e. obtained directly from the chased animals) were reduced to single cell suspensions and plated again (unsorted) in matrigel/serum-free culture conditions. The corresponding secondary organoids were then dissociated and plated to obtain tertiary ones (B). Using limiting dilutions, individual GFP⁺ cells were cultured for 0 to 20 days in Matrigel under serum-free conditions, thus forming a polarized epithelial organoid. The bars represent 50 μM (C).</p

MPA induced regulation of TGF-β and Wnt/β-catenin signaling in the IKPRAB-36 cell line.

<p>A and B: In these pathways a green color represents down regulation by MPA and a red color represents up regulation by MPA. Signaling pathways were provided by Ingenuity Pathway Assist Software© and individual gene expression levels are available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030840#pone.0030840.s004" target="_blank">Table S4</a>. C: Western blot showing FOXO1 expression in the IKPRA-1, IKPRB-1, IKPRAB-36 and IKLV-8 cell lines cultured in the absence (control) or presence (MPA) of 1 nM MPA. *indicates significant regulation in the micro-array analysis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030840#pone.0030840.s004" target="_blank">Table S4</a>).</p

RT-PCR results of genes of interest in the patient samples.

<p>CXCL14, DKK1, DKK4, WIF1 and PEG10 were selected from the micro-array results and verified with real time RT-PCR. Significance was calculated using a Mann-Whitney U-test. A p-value of 0.05 was considered to be statistically significant.</p

Progesterone induced inhibition of migration in a wound-healing assay.

<p>IKPRA-1 (A), IKPRB-1 (B) and IKPRAB-36 (C) cells were cultured in the absence (white bullets) or presence (black bullets) of 1 nM MPA and used for a wound-healing assay (n = 3) and closure of the wound was measured as a percentage of total closure (100% means the wound is open, 0% means the wound has closed). D shows representative images of the process of wound-healing with in red the wound. E shows IF for nuclei (DAPI) and vimentin expression on the invasive front of the manually inflicted wound. In this figure, the wound was always situated on the right side.</p

Expression and histological distribution of PRA+PRB and CD4+, CD8+ and Foxp3+ T-lymphocytes in primary endometrial carcinoma specimens.

<p>A: Overview of immunohistochemical staining for CD4, CD8 and FOXP3 in primary endometrial cancer specimens in non-progressive disease (n = 9) compared to progressive disease (n = 9) (magnification 0,4x, inlay 10x). Non-progressive disease shows pronounced staining, whereas progressive disease shows reduced staining. The scale-bar represents 10 mm. B: Quantification of CD4, CD8 and FOXP3 cell counts on the tumor edge (Tumor Edge), in the tumor (Intratumoral) and on the endometrial-myometrial border (EM border). *indicates a p-value<0.05 (Mann-Whitney U-test). C and D: Representative non-progressive (C) and progressive (D) patient tissues were stained for CD4, CD8 and PRA+PRB and show a positive correlation between the presence of TILs and the expression of PR. Magnification is 5x and the scale-bar represents 1 mm. Patients 6 and 11 were both included in the micro-array analyses. Furthermore patient 11 had only recurrent disease, while patient 12 had recurrent and metastatic disease.</p