Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Delayed endometrial decidualisation in polycystic ovary syndrome; the role of AR-MAGEA11

doi: 10.1007/s00109-019-01809-6

Figure Lengend Snippet: Patient demographics

Article Snippet: DHT-treated PCOS ESC cells were then fixed using 1% formaldehyde solution (Sigma®), quenched with 2.5 M Glycine (Sigma®), and centrifuged following Active Motif’s Epigenetic Services ChIP Cell Fixation Protocol instructions.

Techniques:

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Delayed endometrial decidualisation in polycystic ovary syndrome; the role of AR-MAGEA11

doi: 10.1007/s00109-019-01809-6

Figure Lengend Snippet: Regulation of MAGEA11 expression in ESC during in vitro decidualisation. Endometrial stromal cells (ESC) isolated from biopsies of fertile ( n = 8) and PCOS ( n = 9) patients were left untreated (C), incubated with cyclic AMP (cAMP, 0.5 mM), dihydrotestosterone (DHT, 10 −6 M) or a combination of both for 24 to 72 h. ESCs were obtained from cells in the secretory phase of the cycle. Cell morphology was recorded throughout the experiment to document decidual changes. a , b ESC shape changes were assessed as described in the “ ” section. ImageJ software was used to calculate the circularity of the cells ( n = 20/group), with a perfect circle having a value of one, while a straight line has a circularity value of zero. c , d Cell media was extracted after ESCs were isolated and treated for 48 h. ELISA experiments were carried out on the conditioned media to establish the concentration of key proteins (prolactin/IGFBP-1) at the secreted level. Values were compared with their corresponding untreated cells. e , f After incubation, cell pellets were collected and RNA extracted to analyse MAGEA11 transcript levels by qRT-PCR. All values represent the average and standard deviation. Statistical analysis of the data was performed between samples of the same clinical sub-group using an ANOVA test followed by a t test (treated vs untreated). * p ≤ 0.05 and ** p ≤ 0.01 were considered significant

Article Snippet: DHT-treated PCOS ESC cells were then fixed using 1% formaldehyde solution (Sigma®), quenched with 2.5 M Glycine (Sigma®), and centrifuged following Active Motif’s Epigenetic Services ChIP Cell Fixation Protocol instructions.

Techniques: Expressing, In Vitro, Isolation, Incubation, Software, Enzyme-linked Immunosorbent Assay, Concentration Assay, Quantitative RT-PCR, Standard Deviation

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Delayed endometrial decidualisation in polycystic ovary syndrome; the role of AR-MAGEA11

doi: 10.1007/s00109-019-01809-6

Figure Lengend Snippet: MAGEA11 expression in proliferative phase endometrium. Proliferative phase endometrium from fertile, anovulatory PCOS and ovulatory PCOS patients was analysed for the expression of MAGEA11/AR as described in the “ ” section. a Immunohistochemistry (IHC) figures showing MAGEA11/AR expression; IHC images display × 10 magnification. Scale = 50 μm. b , d Analysis of the IHC scores (H-score) for MAGEA11 and AR antibodies. Fertile ( n = 18); anovulatory PCOS (anovPCOS, n = 21); ovulatory PCOS (ovPCOS, n = 25); E, epithelial; S, stromal. c , e RNA samples obtained from endometrial biopsies were analysed for the expression of MAGEA11/AR transcript levels as described in the “ ” section. Fertile ( n = 10); anovPCOS ; ovPCOS ( n = 6). All values represent median and inter-quartile ranges (box and whisker). Statistical analyses were performed using the Mann-Whitney U test. * p ≤ 0.05 and ** p ≤ 0.01 were considered significant

Article Snippet: DHT-treated PCOS ESC cells were then fixed using 1% formaldehyde solution (Sigma®), quenched with 2.5 M Glycine (Sigma®), and centrifuged following Active Motif’s Epigenetic Services ChIP Cell Fixation Protocol instructions.

Techniques: Expressing, Immunohistochemistry, Whisker Assay, MANN-WHITNEY

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Delayed endometrial decidualisation in polycystic ovary syndrome; the role of AR-MAGEA11

doi: 10.1007/s00109-019-01809-6

Figure Lengend Snippet: MAGEA11 expression in secretory phase endometrium. Secretory phase endometrium from fertile ( n = 33) and ovulatory PCOS (ovPCOS, n = 19) patients was analysed for the expression of MAGEA11/AR as described in the “ ” section. a Inmunohistochemistry (IHC) figures showing MAGEA11/AR expression; IHC images display × 10 magnification. Scale = 50 μm. b , d Analysis of the IHC scores (H-score) for MAGEA11 and AR antibodies. Fertile ( n = 33); ovPCOS ( n = 19); E, epithelial; S, stromal. c , e RNA samples obtained from endometrial biopsies were analysed for the expression of MAGEA11/AR transcript levels as described in the “ ” section. Fertile ( n = 12); ovPCOS ( n = 8). All values represent the median and inter-quartile range (box and whisker). Statistical analysis was performed using the Mann-Whitney U test. * p ≤ 0.05 and ** p ≤ 0.01 are considered significant

Article Snippet: DHT-treated PCOS ESC cells were then fixed using 1% formaldehyde solution (Sigma®), quenched with 2.5 M Glycine (Sigma®), and centrifuged following Active Motif’s Epigenetic Services ChIP Cell Fixation Protocol instructions.

Techniques: Expressing, Whisker Assay, MANN-WHITNEY

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Delayed endometrial decidualisation in polycystic ovary syndrome; the role of AR-MAGEA11

doi: 10.1007/s00109-019-01809-6

Figure Lengend Snippet: MAGEA11 protein localises to the nucleus of endometrial stromal cells and interacts with AR in PCOS patients. a Immunofluorescence confocal microscopy images of fertile and PCOS hESCs that were either left untreated (control, left panel) or treated with DHT for 48 h (DHT, right panel). ESCs were obtained from cells in the secretory phase of the cycle. Fixed cells were incubated with anti-AR and anti-MAGEA11 antibodies for detection. Scale = 20 μm. b Cell lysates from fertile ( n = 3) and PCOS ( n = 3) ESCs were subjected to western blot analysis. The left panel shows a representative blot of MAGEA11 protein expression and the right panel displays the corresponding densitometry; values were normalised to GAPDH expression and are expressed as average and standard deviation (SD). The statistical analysis was performed using Student’s t test; * p ≤ 0.05 is considered significant. c Cell lysates from PCOS ESCs were either left untreated (control, left panel) or treated with DHT for 24 h (right panel) and subjected to protein co-immuneprecipitation using an anti-AR antibody (N-20). Input was used as positive control and IgG as a negative control

Article Snippet: DHT-treated PCOS ESC cells were then fixed using 1% formaldehyde solution (Sigma®), quenched with 2.5 M Glycine (Sigma®), and centrifuged following Active Motif’s Epigenetic Services ChIP Cell Fixation Protocol instructions.

Techniques: Immunofluorescence, Confocal Microscopy, Control, Incubation, Western Blot, Expressing, Standard Deviation, Positive Control, Negative Control

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Delayed endometrial decidualisation in polycystic ovary syndrome; the role of AR-MAGEA11

doi: 10.1007/s00109-019-01809-6

Figure Lengend Snippet: Top androgen receptor targets in endometrial (PCOS) and castrate-resistant prostate cancer tissues

Article Snippet: DHT-treated PCOS ESC cells were then fixed using 1% formaldehyde solution (Sigma®), quenched with 2.5 M Glycine (Sigma®), and centrifuged following Active Motif’s Epigenetic Services ChIP Cell Fixation Protocol instructions.

Techniques:

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Delayed endometrial decidualisation in polycystic ovary syndrome; the role of AR-MAGEA11

doi: 10.1007/s00109-019-01809-6

Figure Lengend Snippet: Genome-wide analysis of AR targeting in PCOS stromal cells. a Diagram showing the average distribution of AR binding sites surrounding gene transcription starting sites (TSSs) in human PCOS tissue. This diagram was generated using the Genomic Regions Enrichment of Annotations Tool (GREAT). Blue columns represent the whole set of filtered AR peaks and green columns represent AR peaks directly linked to the PCOS disease, according to GREAT databases. b Sequence logos and correspondent p values of transcription factor binding motifs enriched for AR in human PCOS tissue, generated with the MEME-Suite application. c Venn diagram showing the overlap between AR binding sites identified in human PCOS tissue and castrate-resistant prostate cancer (CRPC) . d Table containing the list of genes identified as targets of AR in PCOS tissue, which are also involved in endometrial cancer disease, according to the NCBI databases

Article Snippet: DHT-treated PCOS ESC cells were then fixed using 1% formaldehyde solution (Sigma®), quenched with 2.5 M Glycine (Sigma®), and centrifuged following Active Motif’s Epigenetic Services ChIP Cell Fixation Protocol instructions.

Techniques: Genome Wide, Binding Assay, Generated, Sequencing